Multiplexed bead arrays for proteomics

ABSTRACT

Bead arrays suitable for analysis by mass spectrometry are disclosed. In an embodiment, a bead array includes multiple reactive sites, each of the reactive sites being capable of binding multiple distinct target analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/125,164, inventor Vladislav B. Bergo, filed Sep. 7, 2018,which, in turn, claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 62/555,235, inventor Vladislav B.Bergo, filed Sep. 7, 2017, the disclosures of both of which areincorporated herein by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberGM103348 awarded by the National Institutes of Health (NIH) and grantnumber 1456224 awarded by the National Science Foundation (NSF). Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 12, 2022, isnamed 84025ACON_SL.txt and is 14,677 bytes in size.

FIELD

The embodiments disclosed herein relate generally to the field of beadarrays and more specifically to the field of encoding beads in beadarrays. The embodiments disclosed herein also relate to the fields ofbead-based analytical assays, proteomics, protein quantification,affinity separations and mass spectrometry.

BACKGROUND

A biological array is a multiplexed analytical platform technology thathas found multiple applications in Life Sciences, particularly in thefields of genomics, metabolomics, lipidomics, glycomics, proteomics,histology and cytology. A biological array usually features a largenumber of distinct capture agents immobilized on a solid support, suchas a glass microscope slide or beads. A capture agent is capable ofbinding a target analyte. Depending upon the chosen type of solidsupport, biological arrays can be classified either as printed arrays orbead arrays. A major difference between printed arrays and bead arraysis that bead arrays often lack positional encoding. Consequently, theidentity of a capture agent, which is bound to a specific bead, may notbe inferred from the spatial location of the bead and other means ofbead encoding must be utilized.

Various methods of encoding bead arrays are known in the art. Suchmethods utilize optical labels, fluorescent labels, optical barcodes,identifier binding ligands and mass tags.

Certain types of bead arrays do not require bead encoding. For example,it may be possible to directly analyze a capture agent by massspectrometry and identify such capture agent based on its measuredmolecular weight, digestion profile or MS-MS fragmentation profile.

It may be also possible to directly analyze a target analyte by massspectrometry and identify such target analyte based on its measuredmolecular weight, digestion profile or MS-MS fragmentation profile.

U.S. Pat. No. 7,846,748 entitled “Methods of quantitation andidentification of peptides and proteins” discloses using Matrix-AssistedLaser Desorption Ionization Mass Spectrometry (MALDI MS) to analyze amixture of unlabeled and isotopically labeled peptides bound to a singlebead. It is disclosed that the amino acid sequence of a bead-boundpeptide may be determined by measuring the peptide fragmentation profileusing tandem MALDI mass spectrometry (MALDI MS-MS) followed by databasesearching or by performing de novo identification. The possibility ofquantitative measurement of peptide abundance is also disclosed, whichrequires the use of isotope labeled peptides, namely peptides containing²H and ¹⁸O isotopes.

U.S. Pat. No. 7,033,754 entitled “Decoding of array sensors withmicrospheres” discloses the use of mass spectrometry to identify anidentifier binding ligand (IBL) bound to a microsphere, which alreadycontains a bioactive agent. In the disclosed approach the IBL functionssimilarly to a bead mass tag. It is also disclosed that thecharacterization of the bioactive agent may be performed directly byusing mass spectroscopy.

U.S. Pat. No. 9,618,520 entitled “Microarray compositions and methods oftheir use”, the entirety of which is incorporated herein by reference,discloses that microarrays may possess no conventional means of encodingthe active agent. Consequently, an active agent may serve as its owncode and the microarray decoding procedure may comprise releasing theactive agent from its corresponding bead followed by identification ofthe active agent using mass spectrometry.

U.S. patent application Ser. No. 13/369,939, Publication No. US2012-0202709 A1, the entirety of which is incorporated herein byreference, discloses providing information about molecular weights ofcompounds that are present or may be present within a bead array. Suchcompounds may include capture agents, targets, probes, secondary probes,linkers and bead mass tags. It is disclosed that the molecular weightinformation may be used to guide acquisition of mass spectrometric dataand also to identify analytes present on individual beads.

An article entitled “MALDI Immunoscreening (MiSCREEN): A Method forSelection of Anti-peptide Monoclonal Antibodies For Use inImmunoproteomics” (Razawi M et al, J. Immunol. Methods 2011, 364, 50-64)discloses anti-peptide antibodies suitable for immunoaffinityenrichment. It also discloses a table containing a list of proteintargets, corresponding tryptic peptides, their amino acid sequences andpredicted molecular weights.

An article entitled “Precision of Heavy—Light Peptide Ratios Measured byMALDI-TOF Mass Spectrometry” (Anderson N L et al, J. Proteome Res. 2012,11, 1868-1878) discloses a MALDI TOF mass spectrum recorded afterincubation of digested human plasma with an anti-peptide antibodyconjugated to protein G Dynabeads. Multiple (over 20) distinct peakswere detected in the 1,000-1,500 m/z region of the mass spectrum.Several of these peaks were assigned to peptides that were specificallycaptured by the bead-conjugated antibody. Another peak was assigned to aproteolytic peptide derived from human serum albumin, which wasnon-specifically bound to the antibody-conjugated bead. The majority ofpeaks were not assigned to a particular analyte and their originsremained unknown.

The prior art does not teach or suggest target-encoded bead arrays, inwhich a reactive site is encoded solely by molecular weights of targetanalytes that specifically bind to a capture agent of the reactive site.In particular, the prior art does not disclose target-encoded beadarrays in which a capture agent of a reactive site is capable of bindingtwo, three, four or a greater number of distinct target analytes and thereactive site is encoded by a combination of two, three, four or agreater number of values, each of the values being derived from amolecular weight of a target analyte that specifically binds to thecapture agent of the reactive site.

Therefore, there is still a need for methods and compositions that wouldenable analysis of bead arrays.

SUMMARY

In one aspect, the present specification describes a bead array thatincludes at least a first reactive site and a second reactive site. Thefirst reactive site includes a first bead and a first capture agent thatis associated with the first bead. The first capture agent specificallyrecognizes a naturally occurring epitope and specifically binds at leasttwo distinct targets that contain such epitope. The distinct targets maybe proteinaceous compounds that have distinct molecular weights. Thedistinct targets may be proteolytic fragments of a single precursorprotein or proteolytic fragments of distinct precursor proteins. Thesecond reactive site includes a second bead and a second capture agentthat is chemically distinct from the first capture agent, is associatedwith the second bead and specifically recognizes an epitope that is notrecognized by the capture agent of the first reactive site. Each of thefirst and the second reactive sites is associated with a uniquecombination that includes at least two distinct values, which arederived from the molecular weights of targets that specifically bind tothe corresponding reactive site. The combination associated with thefirst reactive site is distinct from the combination associated with thesecond reactive site.

In another aspect, the present specification describes a method ofperforming affinity binding using a bead array, which includes the stepsof contacting a bead array with a sample, binding a first target and asecond target from the sample to a first reactive site of the bead arrayand binding a third target from the sample to a second reactive site ofthe bead array. The first and the second reactive sites contain a beadand a capture agent, the capture agent of the first reactive site beingdifferent from the capture agent of the second reactive site. The firsttarget and the second target are proteinaceous compounds that havemolecular weights less than 5000 Da and contain a naturally occurringepitope, which is recognized by a capture agent of the first reactivesite. The third target contains an epitope, which is recognized by acapture agent of the second reactive site and not recognized by thecapture agent of the first reactive site.

In yet another aspect, the present specification describes a method ofdecoding a bead array, which includes the steps of receiving a massspectrum that was produced by analyzing a reactive site of themicroarray, detecting a first signal and a second signal in the massspectrum and verifying that m/z values of the first signal and of thesecond signal or equivalents thereof match values, which are present ina combination associated with at least one of the reactive sites of themicroarray.

In yet another aspect, the present specification describes a method ofmaking a bead array. The method includes the step of evaluatingindividual reactive sites of a bead array to determine the identity andmolecular weights of target analytes, which may bind to each reactivesite, either specifically or non-specifically. The method may optionallyinclude the step of entering information about the identity andmolecular weights of the target analytes into a decoding table, which isthen included with the bead array.

In yet another aspect, the present specification describes severalmethods of identifying a target analyte within a bead array. Some of thedescribed methods involve measuring a value derived from a molecularweight of the target analyte and subsequently using a decoding tableprovided with the microarray to determine the identity of the targetanalyte. The described methods may be utilized to analyze various typesof biological samples, including cell free proteintranscription-translation reactions, bacterial cell cultures, mammaliancell cultures, cell culture supernatants, animal models, xenografts,tissue biopsies, biofluids and others.

The analytical methods and compositions described in this specificationmay be utilized in a broad range of applications including basicresearch, pharmaceutical drug discovery and drug development, diseasediagnostics and prognostics, biomarker discovery and validation,personalized medicine, precision medicine, systems biology and others.

DESCRIPTION OF FIGURES

The presently disclosed embodiments will be further explained withreference to the attached drawings, wherein like structures are referredto by like numerals throughout the several views. The drawings shown arenot necessarily to scale, with emphasis instead generally being placedupon illustrating the principles of the presently disclosed embodiments.

FIG. 1A schematically depicts a bead array that includes severaldistinct reactive sites. Each of the reactive sites includes a bead andmultiple copies of a capture agent capable of specifically binding twoor more distinct target analytes.

FIG. 1B schematically depicts a decoding table that includes severalentries containing information about target analytes, which specificallybind to individual reactive sites of the bead array. The informationabout target analytes includes their identity, such as name, database IDand/or sequence and their molecular weights, mass-to-charge ratiosand/or time-of-flight values.

FIG. 2A schematically depicts a method for performing affinity binding,in which distinct proteolytic fragments of a precursor protein arecaptured on distinct reactive sites of a bead array.

FIG. 2B schematically depicts a method for performing affinity binding,in which proteolytic fragments derived from distinct precursor proteinsare captured on a same reactive site of a bead array.

FIG. 3 is a photograph of a suspension bead array containing multipledistinct reactive sites inside a microcentrifuge tube placed on amagnetic bead separation rack.

FIG. 4 is a photograph of a microwell slide attached to the 8-wellPROPLATE® module and positioned in a middle section of the PROPLATE®tray for spring modules.

FIG. 5 is a photograph of a bead array, in which 400 μm magnetic agarosebeads are arrayed inside 500 μm wells on a microwell slide.

FIG. 6A is a bright field microscope image of an array of 500 μmdiameter microspots containing crystals of CHCA MALDI matrix, with somemicrospots also containing agarose beads that were reduced in size dueto desiccation.

FIG. 6B is a bright field microscope image of an array of 500 μmdiameter microspots containing crystals of CHCA MALDI matrix afterremoval of the agarose beads.

FIG. 7 shows exemplary mass spectra recorded from individual reactivesites of 4-plex bead array after exposing the array to enzymaticallydigested MKN-45 cell lysate.

DETAILED DESCRIPTION

The terms “microarray” and “bead array” are used interchangeablythroughout the instant specification and refer to a group that includesat least two reactive sites.

The term “reactive site” refers to a combination of a bead and a captureagent that is associated with the bead.

The term “capture agent” refers to a molecule or a molecular complexthat is capable of specifically binding a compound. A non-limitingexample of a capture agent is a monoclonal antibody. A singular form ofthe term “capture agent” may refer to a plurality of molecules or aplurality of molecular complexes. For example it may refer to aplurality of antibody molecules.

The terms “target analyte” and “target” are used interchangeablythroughout the instant specification and generally refer to a bindingpartner of a capture agent. A non-limiting example of a target analyteis a peptide. Singular forms of the terms “target analyte” and “target”may refer to a plurality of molecules, e.g. a plurality of peptidemolecules.

The terms “peptide” and “polypeptide” are used interchangeablythroughout the instant specification and refer to a compound thatcontains at least two amino acids linked by an amide bond, which is alsoknown as a peptide bond.

The term “protein” has the same meaning as commonly used in the fieldsof biochemistry, biophysics and molecular biology. It generally refersto a molecule or a molecular complex that contains at least onepolypeptide.

The term “proteinaceous compound” encompasses a peptide, a polypeptideand a protein.

The terms “well” and “microwell” are used interchangeably throughout theinstant specification and refer to a topological feature such as a pitor a depression that is able to hold a liquid medium, a particle orboth.

The term “value” is commonly defined as a numerical amount denoted by analgebraic term; a magnitude, quantity, or number.

In an embodiment, as schematically depicted in FIG. 1A, the instantspecification describes a microarray, i.e. a bead array that includes afirst reactive site 102 and a second reactive site 103. The firstreactive site includes multiple copies of a first capture agent 112bound to a first bead 111. The first capture agent 112 is configured tospecifically bind a first target 121 and a second target 122. Both thefirst target and the second target are proteinaceous compoundscontaining an epitope 128, which is specifically recognized by the firstcapture agent. The molecular weight of the first target and themolecular weight of the second target are less than 5000 Daltons (Da).The molecular weight of the first target is different from the molecularweight of the second target. The molecular weight of the first targetmay differ from the molecular weight of the second target by less than 1Da or by as much as several thousand Da. In an embodiment, a differencebetween the molecular weight of the first target and the molecularweight of the second target is greater than 5 Da. For example, themolecular weight difference between the first target and the secondtarget may be greater than 10 Da, greater than 20 Da, greater than 50Da, greater than 100 Da, greater than 200 Da, greater than 500 Da orgreater than 1,000 Da. The second reactive site includes multiple copiesof a second capture agent 114 bound to a second bead 113. The secondcapture agent is different from the first capture agent and configuredto specifically bind a first target 123, a second target 124 and a thirdtarget 125. The capture agent of the second reactive site specificallyrecognizes an epitope 129, which is not recognized by the capture agentof the first reactive site. Each of the three targets 123, 124 and 125has a distinct molecular weight Each of the three targets 123, 124 and125 contains the epitope 129. The first and the second beads may bemanufactured from a biocompatible material, such as glass, polystyrene,polypropylene, other types of polymers, agarose, cellulose, other typesof hydrogels or a composite material. Each of the first and the secondcapture agents may be a monoclonal antibody, a polyclonal antibody, anantibody fragment, a single domain antibody, an aptamer, a SOMAmer®reagent, an affimer, a protein, a polypeptide, a receptor, a ligand, anenzyme, an enzyme substrate, an enzyme inhibitor or any other compoundcapable of affinity binding. For example, monoclonal or polyclonalantibodies may be selected to recognize a particular epitope such thatan antibody conjugated to the first bead recognizes a different epitopethan an antibody conjugated to the second bead.

In an embodiment, the first and the second beads and the first and thesecond capture agents are unlabeled, that is they do not comprise adetectable label. For example, both the beads and the capture agents maylack optical labels and mass tags. Furthermore, the first and the secondbeads may also lack positional encoding. Microarrays, in which thereactive sites lack positional encoding, do not allow identification ofa capture agent based on a location of the reactive site.

Preferably, the amounts of the capture agents present on the first andthe second beads are sufficient to enable binding of at least 100attomoles of each target to their respective reactive sites. Dependingupon the specific properties of the bead and the nature of the capturereagent, each of the reactive sites may have the capacity to bind morethan 1 picomole or even more than 10 picomoles of their respectivetarget.

The microarray may further include at least one replicate of at leastone reactive site. For example, a replicate of the first reactive siteschematically depicted in FIG. 1A may include a bead and a bead-linkedcapture agent that is chemically indistinguishable from the captureagent of the first reactive site. The microarray may contain between 1and 100 replicates of a particular reactive site. For certain analyticalapplications it may be preferable that a microarray contains arelatively low number of replicates of a particular reactive site. Forexample, a microarray may contain fewer than 20, fewer than 15, fewerthan 10 or fewer than 5 replicates of a particular reactive site. Infact, it is often sufficient to provide 1, 2, 3 or 4 replicates of aparticular reactive site. In one aspect, having a low number ofreplicates of a particular reactive site may help ensure that alow-abundance analyte does not become overly diluted among manyidentical reactive sites, which would lead to a decrease in theintensity of a signal acquired from an individual reactive site. Inanother aspect, having a low number of replicate reactive sites may helplower the cost of the microarray manufacturing by reducing the amount ofreagents, e.g. antibodies needed to make such microarray. In yet anotheraspect, having a low number of replicate reactive sites may helpincrease the multiplexing capability of a particular bead-based assaybecause a greater number of non-replicate reactive sites may be combinedin a smaller reaction volume and subsequently analyzed on a solidsupport. It is noted that the methods and compositions disclosed in theinstant specification enable highly efficient handling and analysis ofindividual beads and minimize the probability of losing beads throughoutthe microarray processing steps. The methods and compositions disclosedin the instant specification may be utilized to prevent losing even asingle bead throughout the microarray processing steps and allow theend-user to analyze every bead within the microarray. The microarrayhandling procedures described in this specification make it possible toassemble a microarray in which at least some reactive sites are unique,i.e. have no replicates within the microarray. This may lead toincreased detection sensitivity for many low abundance analytes.

In an embodiment, as schematically depicted in FIG. 1B, the microarrayfurther includes a decoding table 130. The decoding table includesinformation about identities and molecular weights of targets thatspecifically bind to individual reactive sites of the microarray. Forexample, an entry 131, which is associated with the first reactive siteand schematically depicted as “REACTIVE SITE 1”, contains informationabout an identity and a molecular weight of the first target thatspecifically binds to the first reactive site, schematically depicted as“TARGET 1-1” and “VALUE 1-1” in columns 132 and 133 of the decodingtable, respectively. The entry 131 further contains information about anidentity and a molecular weight of the second target that specificallybinds to the first reactive site, schematically depicted as “TARGET 1-2”and “VALUE 1-2”, respectively. Likewise, an entry associated with thesecond reactive site, schematically depicted as “REACTIVE SITE 2”,contains information about an identity and a molecular weight of each ofthe three distinct targets that specifically bind to the second reactivesite.

If the target is a protein or has been derived from a protein, forexample via enzymatic digestion of a protein, the information about anidentity of the target may be one or more of the following: a name ofthe protein, an identifier of the protein, an accession number of theprotein in a database, an amino acid composition of the protein, anamino acid composition of a region within the protein and an amino acidcomposition of a site within the protein. More generally, theinformation about an identity of a target may include one or more of thefollowing: the name of the target, its chemical composition, itsstructural formula, etc. The information about a molecular weight of atarget may be a value derived from the molecular weight of the target.Specifically, it may be one or more of the following: the molecularweight of the target, a mass-to-charge (m/z) ratio of the target, atime-of-flight (TOF) value of the target. It may also be anotherquantitative value that may be measured by mass spectrometry, such asthe m/z ratio or the TOF value of: (1) a singly- or a multiply-chargedform of the target, (2) a hydrogen adduct, ammonium adduct, sodiumadduct, potassium adduct or other adduct of the target, (3) a targetthat underwent a loss of water, ammonia or other modification resultingin a decrease in its molecular weight, etc, (4) a target that underwentoxidation or other modification resulting in an increase in itsmolecular weight, etc. The decoding table may optionally also containinformation about an identity of the corresponding capture agent. Forexample if the capture agent is an antibody, the decoding table maycontain information about a sequence of the epitope recognized by theantibody, a manufacturer of the antibody, a catalog number of theantibody, etc. The data included in the decoding table may be stored inthe printed medium, e.g. printed on a sheet of paper; alternatively itmay be stored in the electronic medium, e.g. in a computer memory, on aremovable memory device such as a USB memory card, in a cloud-basedstorage, etc. The data included in the decoding table may be alsoavailable via other means, e.g. posted on a website or sent and receivedby electronic communication, e.g. electronic mail or text messaging.Alternatively, the data included in the decoding table may be generatedon-demand by a software program or another form of a computer algorithm.

Providing the decoding table enables identification of a target analytebased on the molecular weight (MW) or other properties of the targetanalyte, which may be measured by mass spectrometry, such as the massover charge (m/z) ratio of the target analyte or the time-of-flight(TOF) value of the target analyte. In an embodiment, a single valuederived from a molecular weight of a target is unambiguously associatedwith an identity of the target and therefore sufficient to unambiguouslyidentify the target within the microarray. In an embodiment, the valuederived from the molecular weight of the target is a value derived fromthe molecular weight of a non-fragmented form of the target. In otherwords, fragmentation of the target by tandem mass spectrometry is notrequired in order to determine the identity of the target. Furthermore,because the microarrays of the instant disclosure do not use liquidchromatography (LC) separations, key analytical parameters associatedwith LC measurements, such as analyte retention times do not need to beincluded in the decoding table.

It is not necessary for the microarray decoding table to containinformation about the identity and the molecular weight of everydistinct target that may specifically bind to a particular reactivesite. In other words, the quantity of distinct targets that mayspecifically bind to a particular reactive site may be greater than thequantity of distinct targets, for which information about the identityand the molecular weight is available in the decoding table. Forexample, in reference to FIGS. 1A and 1B, the first reactive site 102may specifically bind more than 2 distinct targets while the secondreactive site 103 may specifically bind more than 3 distinct targets.The quantity of distinct targets, for which information about theidentity and the molecular weight is available in the microarraydecoding table, should be sufficient to allow unambiguous identificationof a particular target, a particular group of targets or a particularreactive site within the microarray. In an embodiment, the decodingtable contains the identity and molecular weight information for 2, 3,4, 5, 6, 7, 8, 9, 10, 11 or 12 distinct targets, which may specificallybind to a particular reactive site. In an embodiment, the informationabout molecular weights of distinct targets, which may specifically bindto a particular reactive site, contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or 12 distinct values. In certain cases, the molecular weight of atarget within a microarray may be sufficiently unique to allowunambiguous identification of the target based on a single measured MW,m/z or TOF value, which is schematically depicted as “REACTIVE SITE N”,“TARGET N” and “VALUE N” in FIG. 1B.

In certain cases, the microarray decoding table may contain an entry fora particular reactive site, in which the quantity of targets, for whichthe molecular weight information is provided, is different from thequantity of targets, for which the identity information is provided. Forexample, the quantity of targets, for which the molecular weightinformation is provided, may be greater than the quantity of targets,for which the identity information is provided. Such case isschematically depicted in FIG. 1B as an entry labeled “REACTIVE SITE 3”,in which the identity information is provided for a single target,depicted as “TARGET 3”, yet the molecular weight information is providedfor two distinct targets, depicted as “VALUE 3-1” and “VALUE 3-2”. Thismay occur, for example, when a particular reactive site is capable ofspecifically binding a target, the identity of which is not known.Alternatively, the microarray manufacturer may choose not to disclosethe identity of every target, for which the molecular weight informationis provided in the decoding table.

The disclosed type of microarrays enables unambiguous identification ofa target not only in a case when the molecular weight of a target isunique, but also in a case where the molecular weight of the target isnot unique, i.e. two or more distinct targets within the microarray haveidentical or very similar molecular weights, which may not resolved bymass spectrometry. For example, in reference to FIG. 1A, the molecularweight of target 122, which specifically binds to the first reactivesite 102, may be very close or identical to the molecular weight oftarget 125, which specifically binds to the second reactive site 103. Iftwo distinct analytes have identical or very similar molecular weightsthey may not be distinguished by MALDI TOF mass spectrometry, althoughin some cases they may be distinguished by tandem mass spectrometry,e.g. MALDI TOF-TOF MS or by other types of mass spectrometry. It is alsopossible that a single target is capable of specifically binding to twoor more distinct reactive sites within a microarray. For example, target121 schematically depicted in FIG. 1A, which specifically binds to thefirst reactive site 102, may also specifically bind to the secondreactive site 103. In such cases, a combination of molecular weights,m/z values or TOF values of two, three, four, five or an even greaternumber of distinct targets, which be may captured by a single reactivesite of the microarray and detected together in a single mass spectrum,a so-called spectral signature will be sufficiently unique to enableunambiguous identification of the corresponding target(s) by massspectrometry. The advantages of using two or a greater number of valuesto determine the identity of a particular target analyte and/or aparticular reactive site may include greater confidence in the analyteidentification and a higher degree of multiplexing for microarray-basedanalytical methods.

The disclosed type of microarrays is also suitable for analyticalapplications where the number of distinct targets captured by a samereactive site varies between different samples, e.g. different celllines or a cell line that has been treated with different chemicalcompounds.

It was previously noted that the disclosed microarray encoding systemmay not require fragmentation or sequencing of a target analyte by massspectrometry in order to identify the target analyte within themicroarray. Nevertheless, analyte sequencing by mass spectrometry, e.g.by tandem mass spectrometry may be optionally performed for some or allof the target analytes present in the microarray, either to increase theconfidence in the analyte identification or for purposes other than theanalyte identification. For example, peptides may be analyzed by tandemmass spectrometry to determine the location of a post-translationalmodification within the peptide sequence.

Sequencing by mass spectrometry may be also used specifically for thepurpose of identifying individual target analytes captured by a reactivesite of the microarray. In such case, information about molecularweights and identities of the target analytes that is contained in thedecoding table may be redundant and accordingly, the decoding table maynot need to be supplied with the microarray. The microarray is decodedusing MS-MS sequencing of individual target analytes captured by themicroarray. One potential benefit of such approach is the possibility ofdiscovering and identifying novel analytes that bind to the reactivesites of the microarray. However, this would require access to a moreadvanced MS instrument that is capable of performing MS-MS (MS2)analysis.

Sequencing by mass spectrometry may be also used for quantitativeprofiling of protein abundance changes in multiple samples usingchemical labeling methods such as tandem mass tags (TMT) and isobarictags for relative and absolute quantification (iTRAQ). Experimentalexamples included in this specification describe large diameter, highbinding capacity magnetic agarose beads that are suitable for performingTMT and iTRAQ based protein quantification studies.

In an embodiment, a microarray of the instant disclosure includesmultiple distinct reactive sites and a microarray decoding means, e.g. amicroarray decoding table. Each of the reactive sites of such microarrayis configured to specifically bind between 1 and 30 distinct targets,each target having a distinct molecular weight. Each of the reactivesites includes a bead and a capture agent associated with the bead. Eachof the reactive sites is associated with a combination that containsbetween 1 and 30 distinct values, each of the values being derived froma molecular weight of a target that specifically binds to thecorresponding reactive site. A combination associated with a particularreactive site serves as a code that enables identification of a targetanalyte using a measured molecular weight of the target analyte or anequivalent parameter, e.g. an m/z or a TOF value. Such combination maybe a part of a microarray decoding table, which includes informationabout: (1) an identity and (2) a molecular weight of each of thetargets. The decoding table may further include information about aquantity of distinct targets that may specifically bind to a particularreactive site. It may also include information about abundance ofindividual distinct targets in a biological sample, such as a biofluidor a cell lysate. The abundance information may be provided in aquantitative or a qualitative form. For example, the decoding table maycontain a statement that certain peptide analytes are either not presentor not detectable in a sample that is prepared from a particular cellline.

The microarrays of the instant disclosure may include between 2 and 100distinct reactive sites. Alternatively, the microarrays of the instantdisclosure may include more than 100 distinct reactive sites.Experimental examples of microarrays featuring multiple reactive sitesare provided in this specification.

The microarrays of the instant disclosure may be configured tospecifically bind between 2 and 100 distinct targets, each of thedistinct targets having a distinct molecular weight. Alternatively, themicroarrays of the instant disclosure may be configured to specificallybind between 100 and 500 distinct targets. Alternatively, themicroarrays of the instant disclosure may be configured to specificallybind more than 500 distinct targets. The distinct targets may be derivedfrom a single protein or from multiple distinct proteins. For example, amicroarray may contain distinct capture agents, which specificallyrecognize and bind distinct proteolytic fragments of a naturallyoccurring precursor protein. The number of distinct proteolyticfragments of such precursor protein, which are recognized by the captureagents included in the microarray, may be 2, 3, 4, 5, 6, 7, 8, 9, 10 orgreater than 10. Binding proteolytic fragments that are derived fromvarious regions of a same precursor protein enables analysis of asubstantial portion of the protein primary sequence, up to a completesequence of the protein. Specifically, a microarray may contain distinctcapture agents that bind distinct proteolytic fragments of a precursorprotein, which collectively account for more than 5%, more than 10%,more than 20%, more than 30%, more than 40%, more than 50%, more than60%, more than 70%, more than 80%, more than 90%, more than 95% or 100%of the primary sequence length of the protein. Such distinct captureagents are associated with distinct beads and therefore the distinctproteolytic fragments are captured by distinct reactive sites. Severalexamples of microarrays capable of binding distinct peptides derivedfrom a same protein are provided in this specification.

The microarrays of the instant disclosure may be configured to performanalysis of fewer than 100 distinct targets, between 100 and 1,000distinct targets or more than 1,000 distinct targets. The number ofdistinct targets that may be analyzed using the microarrays of theinstant disclosure is determined by several factors including chemicalcomposition of the individual targets, spectral resolution of the massspectrometer and the detection mass range of the mass spectrometer. Forexample, for proteolytic peptides measured in the linear mode by MALDITOF MS, the detection mass range may be set between approximately 800 Daand 8,000 Da. If molecular weights of the individual proteolyticpeptides are spaced apart by about 5 Da, which is well within thespectral resolution capability of the modern MALDI TOF MS instruments,more than 1,000 distinct target analytes may be measured andunambiguously identified within such microarray.

The microarrays of the instant disclosure may be configured for bindingmultiple distinct targets, each of the distinct targets having amolecular weight that is greater than 700 Da and lower than 30,000 Da.The microarrays of the instant disclosure may be also configured forbinding targets that have a molecular weight lower than 700 Da orgreater than 30,000 Da. Experimental examples of microarrays capable ofbinding targets having molecular weights below 1,000 Da, between1,000-10,000 Da, and greater than 10,000 Da are provided in thisspecification.

The microarrays of the instant disclosure may be configured to performbinding of multiple targets derived from a region of a protein that isadjacent to its C-terminus. To achieve this, an antibody may be selectedto recognize an epitope located sufficiently close to the C-terminus inthe protein primary sequence, e.g. less than 3, less than 5, less than7, less than 10, less than 15, less than 20 or less than 25 amino acidsfrom the protein C-terminus. Such microarrays may be used to detectprotein heterogeneity, proteolytic degradation and/or proteinmodifications in the C-terminal region, among other effects.

The microarrays of the instant disclosure may be configured to performbinding of multiple targets derived from a region of a protein that isadjacent to the N-terminus of the protein. To achieve this, an antibodymay be designed to recognize an epitope located sufficiently close tothe protein N-terminus in the primary sequence, e.g. less than 3, lessthan 5, less than 7, less than 10, less than 15, less than 20 or lessthan 25 amino acids from the protein N-terminus. Such microarrays may beused to detect removal of the initiator Methionine and/or acetylation ofthe protein N-terminus, among other effects.

In an embodiment, the microarrays of the instant disclosure areconfigured for binding multiple distinct targets and include a decodingtable that contains information about a fragmentation pattern of atleast one of the distinct targets. The information about a fragmentationpattern of a particular target may be used to confirm an identity of thetarget. Experimental examples of microarrays that include a decodingtable containing information about a fragmentation pattern of a targetare provided in this specification.

The microarrays of the instant disclosure may include one or severalreactive sites that lack positional, optical and mass tag encoding. Inan embodiment, all reactive sites of a microarray lack positional,optical and mass tag encoding. Furthermore, unlike multiplexed sandwichimmunoassays, the microarrays of the instant disclosure include only asingle (capture) antibody per target analyte and do not require and donot include two distinct antibodies (capture and detection) per targetanalyte.

In an embodiment, the instant specification discloses a microarray thatincludes at least two distinct reactive sites and a decoding table. Eachof the distinct reactive sites is capable of specifically binding atleast one target. The decoding table contains information about anidentity and a molecular weight of the targets that specifically bind toeach of the distinct reactive sites and also information about anabundance of at least one of the targets in a sample. The decoding tablemay also optionally include a description of the sample, for which thetarget abundance data is provided.

Providing information about abundance of a target in a sample may helpincrease confidence in identification of the target by mass spectrometryafter reacting the microarray with the sample.

The abundance information for a particular target may be provided invarious forms. In an embodiment, the abundance information is providedas information about possible presence of the target in a sample, i.e. aparticular target may be expected to be either present or absent in thesample. For example, a protein of mouse origin is not expected to bepresent in a sample derived from a human cell line. In another example,it may be known that a particular protein or a protein isoform is notexpressed in a particular cell line, a particular organ or a particulartissue type and therefore its abundance in such sample will be zero. Inan embodiment, the abundance information is provided as informationabout relative abundance of two or more targets in a sample. Forexample, two isoforms of a particular precursor protein may be expectedto be present in a certain ratio, or at least one of the isoforms may beexpected to be more abundant than the other. In another example, two PTMsites of a particular precursor protein may be expected to be present ina certain ratio, in which one site is modified, e.g. phosphorylated to agreater extent than the other. In an embodiment, the abundanceinformation is provided in quantitative form. For example, informationabout abundance of a particular protein in serum or plasma may be known,at least approximately and expressed as the range of concentrations,e.g. mg/ml, ng/ml or pg/ml. In another example, an approximate intensityof a mass spec signal expected from a particular analyte may beprovided.

The description of the sample, for which the target abundance data isprovided, may include one or more of the following: the sample origin,e.g. a particular cell line, tissue, biofluid, etc; the sample treatmentconditions, such as exposure of a cell line to a particular chemicalcompound; the sample processing history, e.g. the use of a particulardigestive enzyme.

Several non-limiting examples of distinct target analytes thatspecifically bind to distinct reactive sites of a microarray areprovided below and further described in the EXAMPLES section of thespecification.

Peptide isoforms: these are peptides that have closely related but notidentical amino acid sequences. The sequence differences may includeamino acid additions, amino acid deletions, amino acid substitutions andamino acid modifications. The amino acid modifications may bepost-translational modifications (PTMs), such as phosphorylation,acetylation, methylation, ubiquitination, glycosylation, etc. A singlepeptide sequence may contain more than one PTM site and more than onePTM type. For example, protein phosphorylation may occur on multipleneighboring sites within a single peptide sequence.

Peptides with different chemical modifications: a chemical modificationof a peptide usually involves covalent attachment of one or morechemical groups to the peptide, either to a side-chain of an amino acidor to the peptide N- or C-terminal group. A large number of reagentscapable of modifying peptides are known. For example, a peptide may becovalently modified with a compound containing an N-hydroxysuccinimide(NETS) ester or an imidoester, which target primary amines of lysineside-chains and the peptide N-terminus. Specifically, peptides may bedifferentially labeled with fluorescent dyes, such as cyanine dyes CY®3and CY®5, coumarin or coumarin derivatives.

Peptides containing a different number of chemical modifications withinan otherwise identical amino acid sequence: for example, a peptide maybe chemically modified at the N-terminal amine and also at lysine sidechain amine(s). Thus, a peptide with two lysine residues may contain 0,1, 2 or 3 chemically modified amines.

An endogenous peptide and an internal standard: an internal standard isusually spiked, i.e. added into a sample, which already contains anendogenous peptide. The internal standard may be a synthetic peptide,which has a sequence that is distinct from the sequence of theendogenous peptide.

Proteolytic peptides containing missed cleavage sites: digestion of aprecursor protein by a digestive enzyme, such as trypsin, chymotrypsin,endoproteinase GluC, LysC, LysN, Arg-C, pepsin, elastase, etc mayproduce multiple peptides containing 0, 1, 2, 3, 4, 5, 6 or a greaternumber of missed cleavages. Such peptides may have a same epitope butdifferent length and consequently different molecular weight.Furthermore, some digestive enzymes may exhibit non-specific activity,for example certain preparations of trypsin may additionally exhibitchymotrypsin activity. In such scenario a proteolytic peptide maycontain a cleavage site that is not typical or expected for thecorresponding digestive enzyme.

Peptides produced by non-enzymatic hydrolysis of a peptide bond: certaintypes of peptide bonds have inherently low stability and may undergohydrolysis even in the absence of a digestive enzyme. These includehydrolysis of methionine-containing peptide bonds in the presence ofcyanogen bromide, hydrolysis of an aspartic acid-proline peptide bond inthe presence of formic acid and hydrolysis of an asparagine-glycinepeptide bond in the presence of hydroxylamine. A partial hydrolysis of apeptide bond will result in appearance of two or more distinct peptidesof different length.

Peptides originating from different species: certain biologicaltechniques, such as production of tumor xenografts coupled withproteolytic digestion may generate a mixture of peptides that originatefrom different species, e.g. human and mouse. Such peptides may have asame epitope but differ in the amino acid composition outside theepitope.

Peptides derived from proteins that share an identical or similarsequence within an epitope, which is recognized by a capture agent ofthe reactive site. Such proteins may be constituents of a samebiological pathway or distinct biological pathways. In the latter case,a single microarray reactive site may be configured to specifically bindpeptides derived from proteins that are constituents of 2, 3, 4, 5, 6,7, 8, 9, 10 or a greater number of distinct biological pathways. Thus, asingle microarray reactive site may be configured to probe changes in 2,3, 4, 5, 6, 7, 8, 9, 10 or a greater number of distinct biologicalpathways.

FIGS. 2A-2B schematically depict some examples of microarrays andmethods of performing affinity binding using microarrays. In referenceto FIG. 2A, a microarray, i.e. a bead array may contain distinct captureagents that are associated with distinct beads 207, 208 and 209. Thedistinct capture agents specifically recognize distinct epitopes thatare present in a sequence of a naturally occurring protein 201. If theepitopes are preserved after proteolytic digestion, the distinctreactive sites will also specifically recognize and specifically binddistinct proteolytic fragments 202, 203, 204 and 205, which contain thecorresponding epitopes. The microarray may contain multiple distinctcapture agents that specifically recognize multiple distinct proteolyticfragments, which collectively account for more than 10%, more than 20%,more than 30%, more than 40%, more than 50%, more than 60%, more than70%, more than 80%, more than 90%, more than 95%, or 100% of a sequencelength of the protein. For example, two proteolytic fragments of aprotein collectively account for more than 20% of a sequence length ofthe protein if the proteolytic fragments are non-overlapping, eachproteolytic fragment contains more than 10 amino acids and the entireprotein contains less than 100 amino acids.

If overlapping proteolytic fragments 202 and 203 contain the sameepitope, which may occur for example due to incomplete enzymaticdigestion, they will be recognized by the same capture agent andcaptured by the same reactive site 207, as schematically depicted inFIG. 2A.

In reference to FIG. 2B, it is possible that proteolytic digestion oftwo distinct proteins 211 and 212 will generate identical fragments 215,which will be captured by a same reactive site 219 of the bead array.Measuring the abundance of the fragment 215 will provide informationabout the abundance of both precursor proteins 211 and 212 but will notprovide information about the abundance of the individual proteins.Including additional reactive sites 217 and 218, which specificallycapture proteolytic fragments 214 and 216, derived from the proteins 211and 212, respectively, will enable protein-specific quantification.Alternatively, it is possible that the fragments 215 derived from theproteins 211 and 212 have a common epitope but different molecularweights due to differences in their amino acid composition outside ofthe epitope. In such case, the fragments will be distinguished by massspectrometry even when captured on a single reactive site.

The distinct capture agents in the microarray may individually recognizedistinct epitopes within a proteinaceous compound that has a molecularweight less than 5000 Da.

The epitope recognized by a capture agent of the microarray may be anaturally occurring sequence that is present in a human protein and in amouse protein, or more generally, in at least two proteins fromdifferent species. Accordingly, a reactive site of the microarray maybind proteolytic fragments that are derived from both human and mouseproteins. An example of such microarray is provided in thespecification.

A microarray may be designed such that an epitope recognized by acapture agent of a first reactive site naturally occurs in a firstprotein and in a second protein while an epitope recognized by a captureagent of a second reactive site naturally occurs in the first proteinbut not in the second protein. An example of such microarray is providedin the specification.

A microarray may be designed such that epitopes recognized by captureagents of a first and a second reactive sites lack proteolytic cleavagesites that are recognized by trypsin and chymotrypsin. Trypsin usuallyrecognizes proteolytic cleavage sites containing lysine or arginine,except when they are followed by proline or are adjacent to aphosphorylated amino acid, such as phosphoserine, phosphothreonine,phosphotyrosine. In some cases, trypsin may not recognize sitescontaining Lys-Lys, Arg-Arg, Lys-Arg or Arg-Lys sequences. Chymotrypsinusually recognizes sites containing a large hydrophobic amino acid suchas tyrosine, tryptophan, and phenylalanine, although it may alsorecognize sites containing leucine, isoleucine and methionine. Suchmicroarray may be useful for profiling samples produced by enzymaticdigestion using either trypsin or chymotrypsin. An example of suchmicroarray is provided in the specification.

A microarray may be designed such that an epitope recognized by acapture agent of a first reactive site lacks a proteolytic cleavage sitethat is recognized by trypsin while an epitope recognized by a captureagent of a second reactive site contains such proteolytic cleavage site.Such microarray may be useful for profiling samples produced byenzymatic digestion using either trypsin or another protease. An exampleof such microarray is provided in the specification.

A microarray may be designed such that epitopes recognized by captureagents of a first and a second reactive sites lack proteolytic cleavagesites that are recognized by at least two digestive enzymes, e.g. by atleast two of trypsin, chymotrypsin, pepsin, elastase, thermolysin,endoproteinase Arg-C, endoproteinase Glu-C and endoproteinase Asp-N. Anexample of such microarray is provided in the specification.

A microarray may be designed such that a capture agent of a firstreactive site specifically recognizes an epitope that lacks a PTM andspecifically binds a first target and a second target, both of whichcontain the epitope, yet only one of the two targets contains the PTM,which is located outside of the epitope. An example of such microarrayis provided in the specification.

A microarray may be designed such that a capture agent of a firstreactive site specifically recognizes an epitope containing a first PTM,e.g. phosphorylation and specifically binds a first target and a secondtarget, both of which contain the epitope while at least one of thetargets also contains a second PTM, e.g. acetylation, methylation,glycosylation, ubiqutination or sumoylation, which is located outside ofthe epitope. A site of the first PTM may be separated from a site of thesecond PTM by less than 10 amino acids. An example of such microarray isprovided in the specification.

A microarray may be designed such that a capture agent of a firstreactive site specifically recognizes an epitope containing at least 2PTMs and specifically binds a first target and a second target, at leastone of which further contains an additional PTM, which is locatedoutside of the epitope. An example of such microarray is provided in thespecification.

A microarray may be designed such that distinct reactive sites of themicroarray specifically bind at least 3 distinct proteolytic fragmentsof a naturally occurring protein. In an embodiment, each distinctfragment contains at least one PTM. An example of such microarray isprovided in the specification.

A microarray may include capture agents that specifically recognizetheir corresponding epitopes in western blot, ELISA, flow cytometry,immunohistochemistry, immunoprecipitation or immunofluorescence assay.An example of such microarray is provided in the specification.

A microarray may include a capture agent that specifically binds twodistinct targets: a first target, which is detectable by linear modeMALDI TOF MS, but not by reflector mode MALDI TOF MS, and a secondtarget, which is detectable by reflector mode MALDI TOF MS. Targetsdetectable by linear mode MALDI TOF MS include compounds that arelabile, e.g. phosphopeptides, or have high molecular weight, e.g.greater than 10 kDa. Goat Anti-Aconitase 2 antibody described in detailin Example 14 recognizes both intact ACO2 protein (MW 85,425 Da,detectable by linear mode MALDI TOF MS using sinapinic acid as a matrix)and its proteolytic fragment, which includes amino acids 541 through 555(MW 4781.1 Da, detectable by reflector mode MALDI TOF MS usingα-Cyano-4-hydroxycinnamic acid as a matrix).

A microarray may include a capture agent that specifically binds twodistinct targets, which require different matrices for detection byMALDI MS. Examples of such different matrices includeα-Cyano-4-hydroxycinnamic acid (CHCA) and 2,5-Dihydroxybenzoic acid(DHB), CHCA and sinapinic acid (SA), CHCA and2′,6′-Dihydroxyacetophenone (DHAP) and other combinations. An example ofsuch capture agent is Goat Anti-Aconitase 2 antibody described in theprevious paragraph.

In an embodiment, two distinct targets do not comprise an isotopiclabel, i.e. the first target and the second target have similar isotopeabundance. For example, both the first and the second targets may havenatural isotope abundance with respect to every chemical element presentin their structure. For carbon and nitrogen, the natural isotopeabundance is known to be about 98.9% of ¹²C and about 99.6% of ¹⁴N,respectively.

In an embodiment, two distinct targets have different isotope abundance,e.g. the first target, the second target or both the first target andthe second target are isotope labeled. Isotope labeling of a peptide maybe achieved by incorporating one or several stable isotopes, such as ²H,¹³C, ¹⁵N and ¹⁸O. Isotope labeling of a peptide may be also achieved byusing radioactive isotopes. The presence of an isotope label in a singleamino acid usually results in a mass shift of between 4 and 10 Darelative to an unlabeled version of the same amino acid.

It is noted that a mass difference between an unlabeled and anisotope-labeled forms of a proteolytic peptide in a typical bottom-upproteomic assay usually does not exceed 10 Da. In part, this is becausethe most common digestive enzyme, trypsin generally produces peptidescontaining a single Lysine or Arginine residue. The ¹³C, ¹⁵Nisotope-labeled versions of Lysine and Arginine differ from theirunlabeled counterparts by 8 and 10 Da, respectively. Using otherdigestive enzymes, such as chymotrypsin may generate proteolyticpeptides containing several Lysine and/or Arginine residues but may alsoproduce peptides that do not contain these amino acids.

In an embodiment, the reactive site is being configured to enable therelease of the first and the second targets from the bead in a form thatis compatible with analysis of the first and the second targets by massspectrometry. Throughout the present specification, the use of massspectrometry is illustrated primarily using an example of MatrixAssisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry(MALDI TOF MS). However, it is noted that numerous other methods of massspectrometric analysis may be alternatively used, both with respect tothe analyte ionization and the analyte detection. In particular, variousmethods of ElectroSpray Ionization MS (ESI MS) analysis may be used,including methods known as Laser Ablation ElectroSpray Ionization(LAESI) and Desorption ElectroSpray Ionization (DESI) MS.

In an embodiment, the first and the second targets are peptides and theanalysis by mass spectrometry is performed by MALDI TOF MS in the linearmode. Advantages of measuring peptides in the linear mode compared tothe reflector mode may include greater detection sensitivity andminimization of spectral contributions from the analyte fragmentation,such as in-source decay (ISD) and post-source decay (PSD). For example,many phosphopeptides are known to undergo extensive fragmentation insidea mass spectrometer when measured by MALDI TOF MS. However, spectraleffects of their fragmentation are less pronounced in mass spectraacquired in the linear mode compared to mass spectra acquired in thereflector mode. For peptide analysis performed by MALDI TOF MS, thepositive detection mode is more frequently used although the negativedetection mode may be utilized as well. For analytes that have amolecular weight lower than approximately 10,000 Da, measurements byMALDI TOF MS may be performed in the reflector mode. In addition,measurements of certain analytes by MALDI TOF MS may be performed in thetandem (MS-MS) mode.

Mass over charge (m/z) ratio is one of the analyte properties that arecommonly measured by mass spectrometry. Converting the measured m/zratio into the analyte molecular weight (MW) is usually straightforward.For example, peptide analytes measured by MALDI TOF MS in the positivemode are often detected as single-charge protonated ions, in which thecharge (z) is +1 and the mass (m) is equal to the molecular weight ofthe peptide analyte plus the mass of a proton. Therefore, massspectrometry may be used to determine molecular weight of a measuredanalyte. Molecular weight, also termed molecular mass, is usuallyreported in atomic mass units (amu) or Daltons (Da). The presence ofstable isotopes, such as ¹³C and ¹⁵N in the analyte structure leads toappearance of multiple peaks in the analyte mass spectra, which areseparated by about 1 m/z unit. Such multiple peaks are known as theisotopic envelope. Due to the presence of the isotopic envelope, theanalyte molecular mass measured by mass spectrometry may be reportedeither as monoisotopic mass or as average mass.

The microarrays disclosed in this specification may be described astarget-encoded bead arrays, in which an identity of a target analytebound to a particular bead may be determined by direct massspectrometric measurement of that target analyte. A target-encoded beadarray may not require positional, optical or mass tag encoding ofindividual beads. Furthermore, a target-encoded bead array may notrequire mass spectrometric analysis of the capture agents. Consequently,a capture agent may remain bound to a bead even after the correspondingtarget analyte has been released from the bead. Retaining the captureagent on the bead after the release of the target analyte from the beadmay help obtain better quality mass spectra, e.g. mass spectra with alower background and a stronger analyte signal. Alternatively, thecapture agent may be released from a bead in a form that is notnecessarily compatible with analysis by mass spectrometry, e.g. themolecular weight of the capture agent may be outside of a mass detectionrange of the mass spectrometer.

A target-encoded bead array is decoded after the binding of a targetanalyte to its respective capture agent has occurred, e.g. after thebead array has been exposed to a sample and preferably after anunreacted portion of the sample has been removed. It is noted thatanalysis by mass spectrometry comprises measurement of signal intensityat multiple m/z positions (multiple mass channels); therefore a singlemass spectrometric measurement may provide sufficient data to determineboth an identity of a particular target analyte and its abundance in thesample.

It is noted that successful identification of a target analyte in atarget-encoded bead array is dependent upon the presence of a sufficientamount of the target analyte in a sample. If the amount of the targetanalyte in the sample is below the detection limit the target analytewill not be detected or identified. Nevertheless, such negative resultprovides valuable information because it reveals that the particulartarget analyte is either absent in the sample or its abundance in thesample is below a certain threshold.

An exemplary method of fabricating a target-encoded bead array isdisclosed below and described in greater detail in the EXAMPLES sectionof the specification. In an embodiment, the method includes the stepsof: (1) selecting a capture agent that is capable of binding to a beadand when bound to the bead is capable of binding at least a first targetand a second target that have different molecular weights and (2)recording a decoding table, the decoding table containing informationabout an identity of the first target, a value derived from themolecular weight of the first target and a value derived from themolecular weight of the second target.

Step 1: selecting a capture agent. This step involves selecting andacquiring antibodies to be utilized in the bead array. An antibody maybe selected for the purpose of profiling a specific biomarker, profilinga specific type of protein modification, profiling a specific biologicalor cellular pathway, profiling multiple cellular pathways, profilingmolecular changes associated with a specific disease or a specificcellular condition, etc. An antibody may be selected to profile totalprotein abundance, alternatively an antibody may be selected to profilesite-specific protein abundance, such as an extent of a proteinpost-translational modification (PTM) at a specific protein site. Anantibody may be purchased from a commercial vendor or becustom-produced. An antibody may be monoclonal or polyclonal. Anantibody may be raised in various host species, e.g. rabbit, mouse,goat, horse, chicken or it may be synthetically produced. Antibodiesfrom different host species may be utilized within a same bead array. Inan embodiment, the immunogen is a peptide. In an embodiment, theimmunogen is a full-length protein or a biological cell. It is preferredthat the epitope sequence or the sequence of an immunizing peptide for aparticular antibody is known. However, it is also possible to utilizeantibodies for which the epitope sequence or the sequence of animmunizing peptide is not known. An example of the latter is shown inthe EXAMPLES section of the specification.

Obtaining an antibody from a commercial vendor may be advantageousbecause many commercially available antibodies are already provided witha list of applications for which these antibodies have been validated,e.g. western blot (WB), immunoprecipitation (IP), immunofluorescence(IF), immunohistochemistry (IHC), flow cytometry (FC), chromatinimmunoprecipitation (ChIP), enzyme-linked immunosorbent assay (ELISA),sandwich immunoassays, etc. Such information may be valuable because itenables integration of quantitative analysis of a specific proteintarget by mass spectrometry with follow-up studies of the same proteintarget performed using the above-listed applications in cell cultures,tissues, biofluids and animal models.

One consideration when selecting an antibody with a known epitope isthat the epitope should be preserved in the target analyte.Specifically, if a sample to be analyzed is produced by incubation of aprotein with a digestive enzyme the epitope preferably should notcontain a cleavage site of the digestive enzyme. For example, forsamples produced by incubation with trypsin the epitope preferablyshould not contain an internal arginine or lysine. Likewise, for samplesproduced by incubation with Lys-C or Lys-N the epitope preferably shouldnot contain an internal lysine. However, the above requirement is notabsolute since incomplete digestion by a digestive enzyme may preservethe epitope even when a potential cleavage site is present within theepitope. In addition, certain protein modifications may alter thepattern of cleavage sites normally recognized by a digestive enzyme. Forexample, phosphorylation of a tyrosine adjacent to a lysine frequentlyresults in a missed cleavage by trypsin. Another consideration forselecting an antibody with a known epitope is that a linear orcontinuous epitope is generally preferred for performing peptideimmunoaffinity enrichment.

Overall, the total number of distinct capture agents, e.g. distinctantibodies selected for a particular bead array may be fewer than 10,between 10 and 30, between 30 and 100, between 100 and 500, between 500and 1,000, between 1,000 and 10,000 or greater than 10,000. Multiplereplicate beads may be provided for each capture agent, e.g. 2 beads, 3beads, 5 beads, 10 beads or a greater number. For spherical beads thatare approximately 300 μm in diameter, about 25 beads may be packed into1 μL volume, about 2,500 beads may be packed into 100 μL volume andabout 25,000 beads may be packed into 1 ml volume. A microwell platewith dimensions of a standard microscope slide, 25×75 mm may be used toarray over 5,000 of 300 μm diameter beads. A microwell plate withdimensions of a standard microtiter plate, 86×128 mm may be used toarray over 25,000 of 300 μm diameter beads.

Step 2: recording a decoding table. This step involves elucidating theidentity of peptides that may bind to individual antibodies previouslyselected in Step 1. Specifically, for a peptide, which is expected tobind to a particular antibody, an amino acid sequence of the peptideincluding possible post-translational modifications (PTMs) and thepeptide molecular weight are preferably elucidated. Such data may begenerated by analyzing the known epitope sequence(s) for a particularantibody and subsequently determining the peptide amino acid compositionoutside the epitope(s) to account for different sample preparationconditions associated with a particular analytical workflow. Forexample, the peptide length and molecular weight may be affected by theselection of a digestive enzyme used to prepare the sample. The use ofcommon proteomic techniques such as cysteine reduction and alkylationwill also affect the amino acid sequence and molecular weight of theresulting peptides. Furthermore, the nature of the sample that is beinganalyzed may also contribute to the presence of different targetanalytes, for example mammalian cells cultured under conditions thatcause elevated levels of protein phosphorylation are expected to producepeptides with greater numbers of phosphorylated sites.

An alternative method, which is also disclosed in the instantspecification, involves experimental testing and validation ofindividual antibodies performed under defined conditions that can besubsequently documented. Such conditions may include the use of acertain digestive enzyme, use of a certain sample type, e.g. cellculture, use of a certain cell line, use of a certain method of massspectrometry and use of a certain analytical technique, e.g. profilingof protein phosphorylation. In the disclosed method an individualantibody, which is preferably bound to a solid support, e.g. a bead, isexposed to a sample containing the target analyte(s) and animmunoaffinity capture reaction is performed. The target analyte(s) thatbind to the antibody are subsequently released from the solid supportand analyzed by mass spectrometry. The analysis by mass spectrometry mayinclude determination of molecular weights of the individual targetanalytes, as well as determination of the amino acid sequence of theindividual target analytes performed using known fragmentationtechniques such as collision induced dissociation (CID), electrontransfer dissociation (ETD), in source decay (ISD), post source decay(PSD), etc. The procedure disclosed above is then repeated for otherantibodies within the bead array.

An exemplary method of analyzing a biological sample using atarget-encoded bead array is disclosed below and described in greaterdetail in the EXAMPLES section of the specification. In an embodiment,the target-encoded bead array includes at least one reactive site and adecoding table. An example of a reactive site is an antibody bound to abead. The bead-bound antibody is capable of binding at least twodistinct target analytes with different MW: a first target analyte and asecond target analyte. The decoding table includes information about theidentity of the first target analyte, information about the MW, m/zratio and/or TOF value of the first target analyte, optional informationabout the identity of the second target analyte and information aboutthe MW, m/z ratio and/or TOF value of the second target analyte.Therefore, the decoding table contains sufficient information to enableidentification of the first target analyte in a bead array based on thedetection of two signals in a mass spectrum, which are associated withvalues derived from the MW of the first target analyte and the MW of thesecond target analyte. In some cases, detection of even a single signalin a mass spectrum may be sufficient to unambiguously identify the firsttarget analyte. This may happen if the molecular weight of the firsttarget analyte is sufficiently unique, that is no other target analytescaptured by the bead array has the same or very similar molecularweight.

In an embodiment, the method of analyzing a biological sample using atarget-encoded bead array includes the following steps: (1) contacting areactive site of the bead array with a sample that contains or maycontain the first target and the second target under conditions thatallow binding of the first target and the second target to the reactivesite; (2) after the contacting step, acquiring a mass spectrum from thereactive site such that a signal from the first target and a signal fromthe second target are either detectable or actually detected in the massspectrum, (3) identifying the signal from the first target and thesignal from the second target in the mass spectrum using the decodingtable provided with the bead array and (4) measuring intensity of thesignal from the first target and intensity of the signal from the secondtarget.

Step 1: contacting the reactive site with a sample. This step mayinclude performing an immunoaffinity purification (IAP) reaction andsubsequently removing an unreacted portion of the sample from thereactive site. Various methods of performing bead-based IAP reactionsare known in the art. For example, a suspension of beads may be simplymixed with an aqueous solution containing the sample and incubated for aspecific amount of time.

Step 2: acquiring a mass spectrum. This step may include eluting thebound target analyte(s) from the bead onto a spot on a solid support,such as a MALDI target plate and using mass spectrometry to analyze theeluted analyte(s) localized within such spot. The bead may remain on thesolid support; alternatively, the bead may be removed from the solidsupport prior to the measurement by mass spectrometry. When analysis bymass spectrometry involves the use of MALDI MS, the eluted analyteshould be mixed with a MALDI matrix. The intensity of the ionizationlaser beam of a MALDI mass spectrometer should be selected such that asufficiently strong signal from the eluted analyte(s) can be recorded,e.g. a signal is considered to be sufficiently strong if itssignal-to-noise ratio is at least 3:1, preferably at least 10:1, morepreferably at least 30:1, most preferably at least 100:1. The methodsdisclosed in the instant specification allow detection of analyteseluted from a single bead by MALDI TOF MS with a signal-to-noise ratiogreater than 1000:1. It is noted that the intensity of the ionizationlaser beam may require an adjustment when multiple spots containingdifferent analytes are measured.

Step 3: identifying the signal from the first target and the signal fromthe second target. In an embodiment, this step includes searching theacquired mass spectrum for a signal, e.g. a peak with an intensity abovethe background level and once such peak has been detected at a specificm/z value, searching the decoding table to find a target analyteassociated with an m/z value that is identical or sufficiently close tothe m/z value detected in the mass spectrum. This process is thenrepeated if other peaks are present in the mass spectrum. In some cases,identification of the target analyte may be accomplished by detecting asingle peak in the mass spectrum. Alternatively, identification of thetarget analyte may be accomplished by detecting two or more peaks in themass spectrum.

Step 4: measuring intensity of the signal from the first target andintensity of the signal from the second target. This step may beaccomplished by using analytical software to determine intensity of thepeak(s) detected in the mass spectrum. The signal intensity may bereported as the peak height, area under the peak or using other knownformats.

Incubation of a sample with individual reactive sites of the microarraymay result in non-specific binding of one or more compounds to thereactive site. Such compounds may be bound to the bead, alternativelythey may be bound to the capture agent or bound to the linker, e.g.Protein A or Protein G. To alleviate the problem of non-specificbinding, the decoding table may contain information about m/z valuesassociated with such non-specifically bound compounds.

In an embodiment, the first target analyte and the second target analyteare peptides that have been differentially modified. For example, twopeptides may be differentially modified at their N-termini, C-termini orboth. The two peptides may be also differentially modified at specificamino acids, e.g. lysine, cysteine, aspartic acid, glutamic acid,arginine, histidine, etc. For the methods disclosed in the instantspecification suitable peptide modifications include modifications withcompounds that have differential isotope abundance as well asmodifications with compounds that have same, e.g. natural isotopeabundance.

In an embodiment, the first target analyte and the second target analyteare peptides that are not differentially modified. For example, thefirst target analyte may be an endogenous peptide produced by enzymaticdigestion of a biological specimen and the second target analyte may bean internal standard peptide added to the enzymatically digestedbiological specimen containing the first target analyte. In thisexample, the first and the second target analytes may have same, e.g.natural isotope abundance but differ in their amino acid composition,the differences in the amino acid composition being a substitution, amodification, an addition or a deletion of at least one amino acid.

In an embodiment, a sample suitable for analysis by the microarrays ofthe instant disclosure is a digested biospecimen, such as a suspensionor a pellet of cultured biological cells, a biological tissue, abiological fluid or a xenograft. The digestion may be performed usingenzymatic or non-enzymatic compounds. Furthermore, the biospecimen maybe subjected to cell lysis prior to performing the digestion reaction.

In certain applications however, the biological sample does not need tobe lysed and/or digested prior to performing the affinity enrichmentstep. For example, unfractionated serum is known to contain a largenumber of circulating peptides. Another example is analysis of secretedpeptides that are released from cultured cells into the cell culturemedium.

The microarrays disclosed in the instant specification are suitable forprofiling diverse types of protein PTMs such as phosphorylation,acetylation, methylation, glycosylation, ubiquitination, sumoylation andothers. In an embodiment, a microarray reactive site contains anantibody or an aptamer that specifically binds to an epitope and everytarget that is specifically captured by the microarray reactive sitecontains the epitope. In an embodiment, the epitope contains fewer than6 contiguous amino acids, fewer than 5 contiguous amino acids or fewerthan 4 contiguous amino acids. In an embodiment, the epitope isdiscontinuous, that is, at least some of the amino acids, which arerecognized by the corresponding antibody, are not adjacent to eachother. In an embodiment, the epitope contains a first PTM and at leastone of the targets contains a second PTM that is located outside of theepitope. In an embodiment, the epitope does not contain a PTM and atleast one of the targets contains the PTM. In an embodiment, each targetthat binds to the microarray reactive site contains a protein site thatis post-translationally modified in at least one target and lacks a PTMin at least one target. In an embodiment, individual targets captured bythe microarray reactive site are derived from distinct proteins that areconstituents of distinct biological pathways.

The present disclosure is described in the following Examples, which areset forth to aid in the understanding of the disclosure, and should notbe construed to limit in any way the scope of the disclosure as definedin the claims which follow thereafter. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the presentdisclosure, and are not intended to limit the scope of the presentdisclosure nor are they intended to represent that the experiments beloware all or the only experiments performed. Efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,volume, time etc.) but some experimental errors and deviations should beaccounted for.

EXAMPLES Materials and Equipment

N-Hydroxysuccinimide (NHS)-activated magnetic 6% agarose beads werecustom manufactured by Cube Biotech (Plymouth Meeting, Pa.). Thediameter of individual beads was between approximately 250 μm and 500μm, as determined by optical microscopy. These polydisperse beads werefurther separated into fractions with narrower size distributionsincluding fractions containing beads in the <280 μm, 280-355 μm, 355-375μm, 355-400 μm, 375-400 μm, 375-420 μm, 400-420 μm, 400-450 μm and >450μm diameter range. The bead fractionation was accomplished by manuallypassing the bead suspension through a series of polyester mesh filters(ELKO Filtering Co, LLC) until a desired size fraction was produced. Thesize fractionated beads were stored in pure isopropanol (FisherChemical, catalog No. A416-4) until further use.

For certain applications, magnetic agarose beads with varying degrees ofagarose cross-linking were used. It was experimentally determined thatbeads with higher degree of agarose cross-linking when placed into wellsof a 96-well multiwell plate filled with an aqueous medium were able towithstand rigorous shaking of the plate, e.g. at a speed of 800 RPM orgreater for over 12 hours without individual beads breaking apart.

For certain applications, magnetic beads with the agarose contentgreater than 6% were used, e.g. the beads contained either 7.5% or 9%agarose. It was experimentally determined that beads with the agarosecontent greater than 6% generated stronger MS signals after binding andsubsequent elution of various peptide analytes.

For certain applications, larger magnetic agarose beads were used, e.g.beads in the 700-790 μm, 790-910 μm and >910 μm diameter range.

For certain applications, smaller size magnetic agarose beads were used,e.g. beads in the 160-200 μm diameter range.

Indium Tin Oxide (ITO) surface coated glass microscope slides, catalogNo. 237001 were from Bruker Daltonics (Billerica, Mass.). Individualslides have dimensions of 25 mm×75 mm×0.9 mm and are designed to be usedwith the MTP Slide Adapter II for MALDI imaging, available from BrukerDaltonics (catalog No. 235380).

Gold surface-coated glass microscope slides were from Fisher Scientific(catalog No. 12-550-59).

Silicone microwell gaskets, also known as SILICONE ISOLATORS™, werecustom manufactured by Grace Bio-Labs (Bend, Oreg.) using a standardgrade of polydimethylsiloxane (PDMS). Overall dimensions of a siliconegasket were 25 mm×75 mm×0.5 mm. Each gasket contained a square gridarray of 26×88 microwells. An internal diameter of each microwell was0.5 mm with adjacent microwells separated by a distance of 0.3 mm (adistance of 0.8 mm measured as center-to-center). An area ofapproximately 2.5 mm between peripheral wells and the edges of thegasket contained no wells.

For certain applications, silicone gaskets with smaller length and/orwidth were used, including 25 mm×73 mm×0.5 mm, 23 mm×75 mm×0.5 mm and 23mm×73 mm×0.5 mm. A silicone gasket with a smaller width and/or lengthwhen affixed to a 25 mm×75 mm ITO glass slide forms a watertight sealthat has a lower risk of breaking during manual handling because thefingers are less likely to inadvertently pull the gasket from the slidewhen the slide is being held from the edges.

PROPLATE® multi-well chambers in several configurations including 1, 2,3, 4, 8, 16, 24 and 64 chambers were purchased from Grace Bio-Labs.

Protein A+G was purchased from Abcam (catalog No. ab52213). The productdescription posted on the manufacturer's website refers to Protein A+Gas “ . . . a genetically engineered fusion protein that combines the IgGbinding profiles of both Protein A and Protein G”.

Unless noted otherwise, consumables such as microcentrifuge tubes,pipette tips, weigh boats etc, were standard research grade. Unlessnoted otherwise, the reagents such as organic solvents, acids, salts,buffers, detergents, MALDI matrices etc, were standard research gradewith a purity of 99% or higher and used as received from themanufacturer without further purification. Standard lab equipmentincluded a microcentrifuge, a microplate centrifuge, magnetic tuberacks, microtiter plate shaker, vortexer, etc.

Robotic liquid sprayer iMatrixSpray for depositing MALDI matrixsolutions was purchased from Tardo GmbH (Subingen, Switzerland). Thedesign and operation of iMatrixSpray are described in a recentpublication: CHIMIA International Journal for Chemistry (2014), Volume68(3), pp. 146-149.

Experimental Results

Some of the experiments performed using the compositions and methodsdisclosed in this application and the resulting experimental data aredescribed below.

Example 1 Preparing Protein A+G Conjugated Magnetic Agarose Beads

Using the magnetic QuicPick device (Bio-Nobile, catalog No. 24001),between 100 and 125 of size-fractionated Cube Biotech PureCube MagBeadsXXL NETS-activated beads in isopropanol were transferred to a 0.65 mLplastic microcentrifuge tube (Costar, catalog No. 3208) containing about300 μL of isopropanol. The isopropanol was subsequently removed. About640 μL of 1× PBS (Fisher Scientific, catalog No. BP24384) was added tothe tube and the tube was rotated at room temperature for 5 minutes. 80μg of recombinant Protein A+G in 1×PBS was added to the tube. Additionalamount of 1×PBS was added until the tube was full. The tube wasincubated on a tube rotator (Silent Shake Revolver from CrystalIndustries, catalog No. HYQ-1130A) at low speed, 4° C. for 3 hours atminimum, usually overnight. The tube was removed from the rotator andcentrifuged briefly for less than 2 seconds. The supernatant wasremoved. 20 of 10× Tris-Glycine buffer was added to quench the remainingreactive NHS groups. The tube was incubated on a tube rotator at lowspeed at room temperature for 30 minutes. The Tris-Glycine buffer wasremoved and replaced with 300 μL of 1× PBS. The beads were washed bymoving them through the solution using the magnetic separation stand(Promega, catalog No. Z5332). The PBS was removed and the wash step wasrepeated one additional time. 400 μL 1× PBS was added for storage andthe beads were stored at 4° C. Throughout the described procedure carewas taken to avoid directly touching the beads with a pipette tip,applying forceful pipette mixing or excessive vortexing.

Example 2 Preparing Antibody Conjugated Magnetic Agarose Beads

About 20 of Protein A+G conjugated beads prepared as described in theprevious Example were transferred to a clean 0.65 mL microcentrifugetube. The storage solution was removed and 300 μL of 1×PBS was added tothe beads. The beads were washed by moving them through the PBS solutionusing the magnetic separation stand. The PBS solution was removed and 5μg of Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® rabbitmonoclonal antibody, catalog No. 4858 (Cell Signaling Technology,Danvers Mass.) was added to the beads. It was experimentally determinedthat optimal results were achieved when the antibody was provided in amedium that did not contain bovine serum albumin (BSA) or glycerol. Theantibody-bead mixture was incubated on the tube rotator at low speed, 4°C. for 3 hours at minimum, usually overnight. The tube was removed fromthe rotator and centrifuged briefly for less than 2 seconds. The unboundantibody was removed from the beads and 300 μL of 1×PBS was added. Thebeads were washed by moving them through the solution using the magneticseparation stand. The PBS solution was removed and the wash step wasrepeated one additional time. 400 μL of 1×PBS was added for storage andthe beads were stored at 4° C.

Example 3 Cross-Linking an Antibody to Protein A+G Magnetic AgaroseBeads

The antibody-conjugated Protein A+G beads prepared as described in theprevious Example were transferred to a clean microcentrifuge tube bymanual pipetting using a wide orifice pipette tip. The microcentrifugetube was placed on a magnetic stand and the PBS solution transferredwith the beads was removed. 300 μL of freshly prepared 0.2M solution oftriethanolamine (Sigma-Aldrich, catalog No. 90279) at pH 8.2 was addedto the beads, the bead suspension was briefly vortexed then centrifugedand the supernatant removed. The wash with triethanolamine was repeatedonce more. 300 μL of freshly prepared 25 mM solution of dimethylpimelimidate dihydrochloride (DMP, Sigma-Aldrich, catalog No. D8388) in0.2M triethanolamine at pH 8.2 was added to the beads. The beadsuspension was briefly vortexed, then centrifuged. The microcentrifugetube containing the bead suspension was placed on a lab rotator and thebeads were incubated while rotating for 45 minutes at room temperature.The beads were subsequently centrifuged and the supernatant removed. 300μL of freshly prepared 0.1M solution of ethanolamine (Sigma-Aldrich,catalog No. 398136) at pH 8.2 was added. The bead suspension wasvortexed then centrifuged and the supernatant removed. The ethanolaminewash step was repeated once more. The microcentrifuge tube containingthe bead suspension was placed on a lab rotator and the beads wereincubated in the ethanolamine solution for 1 hour at room temperaturewhile rotating. The supernatant was subsequently removed, the beads werewashed twice with 300 μL of 1×PBS buffer (Fisher Scientific, catalog No.BP24384300) and stored in 1×PBS buffer at 4° C.

Example 4 Assembling a Multiplexed Target-Encoded Bead Array

Monoclonal antibodies specific for protein targets involved in themechanistic target of rapamycin (mTOR) and other pathways were purchasedfrom Cell Signaling Technology (Danvers, Mass.), R&D Systems(Minneapolis, Minn.) and ThermoFisher (Waltham, Mass.). The antibodiespurchased from Cell Signaling Technology included Phospho-p70 S6 Kinase(Thr389) (108D2) rabbit mAb (catalog No. 9234), Phospho-p70 S6 Kinase(Thr389) (1A5) mouse mAb (catalog No. 9206), p70 S6 Kinase (49D7) rabbitmAb (catalog No. 2708), Phospho-S6 Ribosomal Protein (Ser235/236)(D57.2.2E) XP® rabbit mAb (catalog No. 4858), Phospho-S6 RibosomalProtein (Ser240/244) (D68F8) XP® rabbit mAb (catalog No. 5364), S6Ribosomal Protein (54D2) mouse mAb (catalog No. 2317), 4E-BP1 (53H11)rabbit mAb (catalog No. 9644), Phospho-4E-BP1 (Thr37/46) (236B4) rabbitmAb (catalog No. 2855), Phospho-4E-BP1 (Ser65) (D9G1Q) rabbit mAb(catalog No. 13443), Phospho-4E-BP1 (Thr70) (D7F61) rabbit mAb (catalogNo. 13396), Phospho-Akt (Pan) (Ser473) (D9E) XP® rabbit mAb (catalog No.4060), Phospho-Akt1 (Ser473) (D7F10) XP® rabbit mAb (catalog No. 9018),Phospho-Akt (Pan) (Thr308) (D25E6) XP® rabbit mAb (catalog No. 13038),Akt (pan) (C67E7) rabbit mAb (catalog No. 4691), Akt1 (C73H10) rabbitmAb (catalog No. 2938), Phospho-TSC2 (Thr1462) (5B12) rabbit mAb(catalog No. 3617), TSC2 (D93F12) XP® rabbit mAb (catalog No. 4308),Phospho-Gsk3b (Ser9) (D85E12) XP® rabbit mAb (catalog No. 5558) andGSK3b (D5C5Z) XP® rabbit mAb (catalog No. 12456). The antibodiespurchased from R&D Systems included Human Phospho-Histone H2AX (Ser139)(catalog No. AF2288), Human/Mouse/Rat Phospho-ATM (Ser1981) (catalog No.AF1655), Human Phospho-BRCA1 (Ser1423) (catalog No. AF1386), HumanPhospho-Chk2 (Thr68) (catalog No. AF1626), Human Phospho-Chk1 (Ser345)(catalog No. AF2475). The total number of unique capture reagents, i.e.antibodies included in the bead array, was greater than 50.

Individual antibodies were conjugated to magnetic agarose beads usingprocedures described in the previous Examples. A series of multiplexedbead arrays for profiling multiple targets in the mTOR pathway wereassembled by selecting between 1 and 3 beads conjugated to one of theantibodies listed above and subsequently combining beads, which wereconjugated to different antibodies, in a single 1.7 mL microcentrifugetube. For example, FIG. 3 shows a multiplexed suspension bead array formeasuring total protein abundance and site-specific phosphorylation oftwo proteins: 4E-BP1 and p70 S6 Kinase. The bead array shown in FIG. 3contained Cell Signaling Technology antibodies #2855, #9644, #2708 and#9234 individually conjugated to 700-790 μm diameter magnetic agarosebeads. The individual reactive sites of this four-plex bead array didnot possess distinguishable optical properties or mass tags nor did theyhave positional encoding. However, target analytes captured by the beadarray were readily identifiable by mass spectrometry because informationabout an identity and a molecular weight of the target analytes wasprovided in the microarray decoding table. The data in the decodingtable was generated by independently validating each of the antibodiesincluded in the bead array. An antibody validation process included thesteps of (1) performing immunoaffinity enrichment of peptide analyte(s)from a digested cell lysate using a single bead conjugated to a specificantibody, (2) using mass spectrometry to measure the analyte(s) capturedby the bead-conjugated antibody and (3) assigning the detected analytesignal(s), i.e. peak(s) in the mass spectra to specific peptidesequence(s). The peak assignment was performed using availableinformation about the precursor protein sequence, the antibodyspecificity, e.g. the probable epitope and the digestive enzymespecificity. Specifically, in this Example all precursor proteins wereof human origin and the digestive enzyme was mass spectrometrysequencing grade trypsin. It was determined experimentally that peptidetargets captured by individual reactive sites of this bead array hadsufficiently unique molecular weights to enable unambiguousidentification of a particular target based on a single value, which wasderived from the molecular weight of the target. Specifically, thereactive site containing antibody #9644 for measuring total proteinabundance of 4E-BP1 captured proteolytic fragment RAGGEESQFEMDI (SEQ IDNO: 1) derived from the C-terminus of 4E-BP1, which contained one missedcleavage site and had an average calculated [MH]+m/z of 1469.58. Thesame reactive site also captured proteolytic fragment AGGEESQFEMDI (SEQID NO: 2), which contained no missed cleavage sites and had an averagecalculated [MH]+m/z of 1313.39. The reactive site containing antibody#2855 for measuring phosphorylation of 4E-BP1 at Thr37 and Thr46captured proteolytic fragment VVLGDGVQLPPGDYSTTPGGTLFSTTPGGTR (SEQ IDNO: 3), which contained two phospho-groups and had an average calculated[MH]+ m/z of 3209.32. The reactive site containing antibody #2855 alsocaptured proteolytic fragment with the same sequence but a singlephospho-group, which had an average calculated [MH]+m/z of 3129.35.

Example 5 Cell Lysis, Protein Digestion and Peptide Extraction

MKN45 human gastric cancer cells were cultured using the RPMI 1640medium supplemented with 20% FBS. Cells grown on a plate to about 80%confluence were washed twice with cold PBS, 1 mL of urea lysis bufferwas added to the plate, the cells were scraped from the plate andcollected in a 50 mL FALCON™ centrifuge tube. The cell suspension wassonicated using a microtip in 3 to 5 bursts for 20 seconds each time at15 W output. The cell suspension was cooled on ice for 1 min between thebursts. The lysate was cleared by centrifugation at 5,000 g for 15 minat room temperature and the supernatant transferred to a clean tube forenzymatic digestion.

The cleared cell supernatant was subjected to reduction, alkylation andenzymatic digestion. 1/278 volume of 1.25M DTT was mixed with thesupernatant and incubated at 40° C. for 30 min. The solution was brieflycooled on ice until it reached room temperature. 1/10 volume of freshlyprepared 100 mM iodoacetamide solution was subsequently added andincubated for 15 min at room temperature in the dark. The solution wasdiluted 5-fold with 0.2M ammonium bicarbonate digestion buffer. 1/100volume of 1 mg/mL sequencing-grade trypsin stock was added and thedigestion reaction allowed to proceed overnight at 37° C. with mixing.For digestion with chymotrypsin, 1/60 volume of 1 mg/mL chymotrypsinstock was added instead of trypsin and the digestion reaction allowed toproceed overnight at 37° C. with mixing.

Immediately after the digestion reaction the digested cell lysate waspurified on 0.7 mL SEP-PAK® C18 columns from Waters Corporation, catalogNo. WAT051910. Prior to loading peptides from the protein digest on thecolumn, the digested lysate was acidified by adding 1/20 volume of 10%TFA to the final concentration of 0.5% TFA. The solution was allowed tostand for 15 min on ice, which resulted in formation of a precipitate.The acidified peptide solution was centrifuged for 15 min at 1,780 g atroom temperature and the peptide-containing supernatant was transferredinto a clean 50 mL conical tube without dislodging the precipitatedmaterial.

A reservoir made from a 10 cc syringe, which had the plunger removed,was connected to the short end of the SEP-PAK® column. 5 mL of 100% MeCNwas applied to pre-wet the column. The column was washed sequentiallywith 1 mL, 3 mL, and 6 mL of 0.1% TFA solution. The acidified andcleared digest was loaded on the column and washed sequentially with 1mL, 3 mL, and 6 mL of 0.1% TFA solution followed by wash with 2 mL of 5%MeCN, 0.1% TFA solution. The peptides were eluted by 3 sequentialwashes, each with 2 mL of 0.1% TFA, 50% acetonitrile solution. Solutioncontaining the eluted peptide was placed inside a −80° C. freezer for atleast 2 hr to overnight. The frozen peptide solution was lyophilizedusing a standard lyophilizer for a minimum of 2 days to assure TFA hasbeen removed from the sample. The lyophilized digested peptides can bekept inside a sealed microcentrifuge tube at −80° C. for several months.

Example 6 Performing Multiplexed Immunoaffinity Enrichment of Peptidesfrom an Enzymatically Digested Cell Lysate Using Antibody-ConjugatedMagnetic Beads

A lyophilized cell lysate prepared as described in the previous Examplewas suspended in the binding buffer (2.5M NaCl in 25 mM Tris, 192 mMglycine, pH ˜8.3) to the final concentration of about 2 mg/mL. Thelysate suspension was homogenized by repeated manual pipetting whileavoiding forming bubbles in the solution. The cell lysate solution wascentrifuged at 14,000 RPM at 4° C. for 5 min. The clear supernatant wastransferred to a new 1.7 mL microcentrifuge tube. Using a wide orificepipette tip, a suspension bead array containing 20 antibody-conjugatedmagnetic beads prepared as described in Example 4 was transferred to aclean 0.65 mL microcentrifuge tube. The clear cell lysate supernatantwas added to the beads while avoiding forming bubbles in the lysate.This was followed by a brief, e.g. less than 1 second centrifugation ofthe tube to collect droplets from the tube walls. The suspension ofbeads mixed with the cell lysate supernatant was incubated on a rotatorat low speed at 4° C. for at least 3 hours, in some cases overnight orlonger than 24 hours. The tube was briefly centrifuged, the beadsremoved using the magnetic QuicPick device and transferred to a clean0.65 mL tube containing 300 μL of 2.5M NaCl solution in deionized water.An additional 340 μL of the 2.5M NaCl solution was added to the tube.The beads were incubated on the rotator at low speed at room temperaturefor 10 minutes. The tube was briefly centrifuged and about 300 μL of thesolution removed by pipetting. Using the magnetic QuicPick device thebeads were transferred to a clean 0.65 mL tube containing 300 μL ofdeionized water. The beads were washed by repeatedly moving the tube onthe magnetic separation stand and further incubated for about 2 minutesat room temperature. Using pipetting with a wide orifice pipette tip,the beads were transferred into a clean 0.65 mL tube containing about200 μL of deionized water and stored at 4° C.

Example 7 Assembling a Microwell Slide

A microwell slide was prepared by affixing a silicone gasket to theconductive side of an ITO-coated glass microscope slide. Specifically, asurface of the silicone gasket was first cleaned from any residual dustparticles by using a strip of adhesive tape. The gasket was then placedon top of one or two sheets of a low lint tissue, such as KIMWIPES®positioned on a flat surface of a laboratory bench. Dry ITO-coatedsurface of the glass slide was placed in contact with the siliconegasket and the slide was manually pressed into the gasket to form awatertight seal. The seal quality was visually checked and any residualair pockets were removed by pressing locally on the slide. Overalldimensions of the fabricated microwell slide were 25 mm×75 mm×1.4 mm.

Example 8 Forming a Bead Array on the Microwell Slide

The microwell slide was prepared as described in the previous Exampleand attached to the PROPLATE® 8-well slide module containing 8rectangular chambers, each of the chambers having internal dimensions of7 mm×16 mm. For certain applications the 1.6 mm thick clear siliconegasket included in the standard PROPLATE® slide module was replaced witha thinner 1.3 mm clear silicone gasket in order to better accommodatethe 1.4 mm thick microwell slide. The microwell slide was secured to thePROPLATE® module by PROPLATE® stainless steel spring clips.

The assembly containing the microwell slide attached to the PROPLATE®module was subsequently placed in the middle section of a three-sectionPROPLATE® tray for spring clip modules, as depicted in FIG. 4 . About300 μL of deionized water was dispensed into each of the 8 chambers andthe PROPLATE® tray was centrifuged at 2700 RPM for 5 minutes at roomtemperature to facilitate entry of water into the 0.5 mm diameter wellsof the microwell slide.

An aqueous suspension containing 42 magnetic agarose beads in the0.375-0.420 mm diameter range was pulled into a wide orifice pipette tipby pipetting and then randomly dispensed into 2 of the 8 chambers of thePROPLATE® module. The PROPLATE® tray was placed on a standard laboratorymicrotiter plate shaker and subjected to mechanical agitation for 1 to 2minutes to help distribute the beads into microwells. The PROPLATE® traywas subsequently centrifuged at 2700 RPM for 1 minute at roomtemperature to help bring the beads down to the bottom of theirrespective microwells. All beads were therefore positioned about 80 to120 microns below the surface of the microwell slide.

The microwell slide attached to the PROPLATE® module was removed fromthe PROPLATE® tray and a 3″×1″×⅛″ thick nickel plated magnetized throughthickness neodymium magnet (K&J Magnetics, catalog No. BZOX02) wasplaced underneath the module between the stainless steel clips to helpretain the beads in their locations. Bulk water was withdrawn from eachchamber by pipetting, with the pipette tip pointing toward a sidewall ofthe chamber so as not to disturb the bead array, the magnet and thestainless steel clips removed and the microwell slide detached from thePROPLATE® module. The microwell slide was placed on top of the magnetand residual drops of water remaining on the surface were removed bygently pressing a sheet of a low lint absorbent tissue, such asKIMWIPES® or alternatively a paper towel against the slide surface. Noneof the beads, which remained positioned about 80 to 120 microns belowthe slide surface, were displaced by this procedure, as seen in FIG. 5 .It was observed that while bulk water was completely removed from thesurface of the microwell slide, individual 0.5 mm diameter microwellsretained sufficient amount of liquid, which remained inside themicrowells for about 5 to 10 minutes under standard conditions, e.g.relative humidity between 10% and 90%, temperature between 18° C. and32° C. As long as some amount of liquid remained in the microwells, theagarose beads retained their fully hydrated size. Only after theresidual liquid had evaporated from the microwells did the beads shrinkto a fraction of their original size as a result of desiccation.

Example 9 Eluting Analytes from a Bead Array

A bead array containing 13 beads was fabricated on a microwell slideusing the procedure described in the previous Example. All individualbeads within the bead array were conjugated to the previously describedPhospho-S6 Ribosomal Protein (Ser235/236) monoclonal antibody #4858 andcontained captured proteolytic fragments of S6 Ribosomal Protein duallyphosphorylated at Ser235 and Ser236.

Droplets of water present on the surface of the microwell slide afterseparation from the PROPLATE® module were removed by gently touching theslide surface with an absorbent tissue. The microwell slide wasimmediately placed at the center of the matrix deposition area ofiMatrixSpray and 10 cycles of matrix deposition were applied to the beadarray. Note that the first matrix deposition cycle should commence nolater than 5 minutes, preferably within 3 minutes after removingdroplets of water from the slide surface in order to achieve optimalmixing of the MALDI matrix solution with the residual liquid remaininginside the microwells. The matrix deposition parameters of iMatrixSpraywere set as follows: Height: 60 mm; Line Distance: 0.5 mm; Speed: 60mm/s; Density: 5 μL/cm²; Number of cycles: 10; Delay: 0 sec; Spray areawidth: 80 mm; Spray area depth: 30 mm. The matrix solution containedα-Cyano-4-hydroxycinnamic acid (CHCA) at 5 mg/mL, 50% acetonitrile(v/v), 0.4% trifluoroacetic acid and 4 mM diammonium citrate.

Once the final matrix deposition cycle was completed, the bead array wasallowed to air-dry, which required about 25 to 30 minutes. The microwellslide was visually examined to verify that no microwells containedresidual liquids and that all beads had been reduced in size due todesiccation. The silicone gasket was separated from the microscopeslide. Some of the dried agarose beads stuck to the silicone and wereremoved from the slide along with the gasket. The remaining beads wereremoved using a stream of compressed air from an air duster, which wasdirected at the slide surface. It was observed that while dried agarosebeads were readily displaced from their positions by the compressed gas,the crystals of MALDI matrix remained firmly attached to the slidesurface and localized within their respective microspots.

FIG. 6A is a bright field microscope image of an array of spotscontaining CHCA MALDI matrix, which was recorded prior to removal of thebeads from the slide by compressed air. Agarose beads, which had beenreduced in size due to desiccation, are visible in 9 spots. Inadditional 4 spots the beads were not present but the spot shape, e.g.non-uniform distribution of CHCA matrix indicated prior presence of abead, which had likely been removed from the slide along with thesilicone gasket. The remaining spots contained no beads and displayedmore uniform distribution of the matrix across the spot. FIG. 6B is abright field microscope image of an unrelated array of spots containingCHCA MALDI matrix, in which all beads had been removed from the slideusing compressed air. Note that spots formed in locations that initiallycontained a bead exhibited characteristic “crescent” or “donut” shapeswith areas containing little or no CHCA matrix. In contrast, spotsformed in locations initially devoid of a bead generally exhibited moreuniform matrix coverage. The images shown in FIG. 6A and FIG. 6B wereacquired on CYTATION™ 3 multi-mode reader from BioTek (Winooski, Vt.).

Example 10 Acquiring Mass Spectrometric Data from an Array of Microspots

An array of microspots on the ITO-coated surface of a microscope slidewas prepared using procedures described in the previous Example. Theslide was placed into the MTP Slide Adapter II for MALDI Imaging. Acustom geometry file provided by Bruker Daltonics (Billerica, Mass.)enabled consecutive acquisition of mass spectrometric data from a squaregrid array of 2288 spots, each spot having a variable diameter of up to600 In some cases, the data was acquired from smaller sections withinthe array, which contained between 50 and 500 spots.

The MALDI TOF MS data was acquired on Bruker Autoflex Speed MALDITOF-TOF mass spectrometer from Bruker Daltonics using flexControlsoftware provided with the instrument. The data was acquired in thelinear positive mode, in 1,000 to 7,000 m/z mass range, using the laserrepetition rate of 2 kHz. A total of 20,000 single-shot spectra weretypically collected from a single microarray spot and co-added, althoughin some cases high quality spectra were obtained from as few as 100shots. In some cases, more than 100,000 single-shot spectra werecollected from a single microarray spot without completely depletinganalytes present in the spot. The spectra were acquired from multiplelocations within each spot using either the random walk or the inversespiral method, both methods provided in the flexControl software.

Example 11 Analyzing Mass Spectrometric Data

MS datasets were analyzed using flexAnalysis and BIOTOOLS™ software fromBruker Daltonics. In some cases, files containing MS data were convertedinto .txt format and analyzed using the public domain software mMass.Mass spectra were analyzed to determine key parameters such as massaccuracy, spectral resolution, as well as signal-to-noise ratio,relative intensity and peak area of individual peaks present in aparticular mass spectrum.

Example 12 Microarray Reactive Site Configured for Capturing MultipleTarget Analytes

In this Example the capture agent is a monoclonal antibody Phospho-Met(Tyr1234/1235) (D26) XP® Rabbit mAb, catalog No. 3077 that was acquiredfrom Cell Signaling Technology (Danvers, Mass.). Met, also known ashepatocyte growth factor receptor (HGFR) is a tyrosine kinase with a MWof 145 kDa. The entry number and the entry name for this protein in theUniProt (Universal Protein Resource) database are P08581 and MET HUMAN,respectively. According to the product description provided on themanufacturer's website, which was accessed on Feb. 20, 2017, #3077antibody “ . . . detects endogenous levels of Met only whenphosphorylated at Tyr1234/1235”. The #3077 antibody is validated forapplications that include western blotting, immunoprecipitation,immunohistochemistry (paraffin), immunohistochemistry (frozen),immunofluorescence (immunocytochemistry) and flow cytometry. Using theprocedures described in the previous Examples, the #3077 antibody wasconjugated to Protein A+G agarose beads and incubated with 1 mg oftryptic digest of lysed MKN-45 cells. A mass spectrum was recorded inthe linear positive mode after performing immunoaffinity purification(IAP) reaction and eluting the bound analytes from a single bead. MSmeasurements were also performed in the reflector positive mode (datanot shown). Three strong peaks were detected in the mass spectrum at m/zvalues of 1853.06, 2210.27 and 2647.54. The assignment of these peaks tospecific peptide analytes was made based on the following data: theknown amino acid sequence of the precursor protein, i.e. Met kinase, theknown specificity of the digestive enzyme, i.e. trypsin and the probableepitope. With respect to the latter, although the precise epitopesequence is not disclosed, the manufacturer's website states that the“monoclonal antibody is produced by immunizing animals with a syntheticphosphopeptide corresponding to residues surrounding Tyr1234/1235 ofhuman Met”. Based on the above information, the peak assignments weremade as follows: the 1853.06 peak was assigned to a peptideDMYDKE[pY][pY]SVHNK (SEQ ID NO: 4), which has a calculated MH+ value of1852.82, the 2210.27 peak was assigned to a peptideDMYDKE[pY][pY]SVHNKTGAK (SEQ ID NO: 5), which has a calculated MH+ valueof 2210.23, and the 2647.54 peak was assigned to a peptideDMYDKE[pY][pY]SVHNKTGAKLPVK (SEQ ID NO: 6), which has a calculated MH+value of 2647.81. In the amino acid sequences listed above [pY] denotesphosphorylated tyrosine. It is noted that the peak assignments did notrequire the peptide fragmentation by mass spectrometry or the use oftandem mass spectrometry (MS-MS). In this Example, the multiple targetanalytes are proteolytic fragments of a precursor protein containingdifferent numbers of missed cleavage sites.

Example 13 Microarray Reactive Site Configured for Capturing a VariableNumber of Target Analytes

In this Example the capture agent is Phospho-S6 Ribosomal Protein(Ser235/236) (D57.2.2E) XP® Rabbit monoclonal antibody, catalog No. 4858that was purchased from Cell Signaling Technology. According to thedescription provided on the manufacturer's website, which was accessedon Feb. 20, 2017, #4858 antibody “ . . . detects endogenous levels ofribosomal protein S6 only when phosphorylated at Ser235 and 236”. Theentry number and the entry name for human S6 ribosomal protein in theUniProt database are P62753 and RS6_HUMAN, respectively. Conjugation ofthe antibody to magnetic agarose beads and immunoaffinity enrichmentreactions were performed using procedures described in the previousExamples. The immunoaffinity enrichment was performed independently fromtwo samples, which were prepared from trypsin-digested lysates of MKN-45cells cultured in the absence and presence of hydrogen peroxide. Theexposure of mammalian cells to hydrogen peroxide is known to causeelevated levels of protein phosphorylation.

Mass spectra of proteolytic peptides captured from the digested lysatesof MKN-45 cells grown in the absence of hydrogen peroxide exhibited atotal of 4 distinct peaks spaced apart by about 80 m/z units. Massspectra of proteolytic peptides captured from the digested lysates ofMKN-45 cells grown in the presence of hydrogen peroxide exhibited atotal of 6 distinct peaks. Four of the peaks were present at identicalm/z positions in the two spectra while the two additional peaks uniqueto the hydrogen peroxide-treated cells appeared at higher m/z and werealso spaced apart by about 80 m/z. Using available information about theprotein sequence, the antibody specificity and the digestive enzymespecificity, all detected peaks were assigned to a proteolytic peptideRLSSLRASTSKSESSQK (SEQ ID NO: 7) consisting of amino acids 233 through249 of human S6 ribosomal protein and containing 3 missed cleavage sitesand a variable number of phosphorylated sites. Predicted average m/zvalues for the differentially phosphorylated peptides were: 2013.1 (2phospho sites), 2093.0 (3 phospho sites), 2173.0 (4 phospho sites),2252.9 (5 phospho sites), 2332.9 (6 phospho sites) and 2412.9 (7 phosphosites). Note that peptides containing no phosphorylated sites or asingle phosphorylated site were not detected because of the specificityof the antibody, which recognizes the peptide sequence the only whenboth Ser235 and Ser236 are phosphorylated. Furthermore, the presence ofmultiple missed cleavage sites in the detected peptide was consistentwith the specificity of this digestive enzyme because it is known thattrypsin may not cleave after Lys or Arg residues proximal to aphosphorylated residue. In this Example, the microarray reactive site iscapable of binding at least 6 distinct peptide analytes, which differ inthe quantity of post-translationally modified sites.

Example 14 Microarray Reactive Site Configured for Capturing ProteolyticPeptides of Human and Mouse Origins

In this Example the capture agent is Goat Anti-Aconitase 2 (aa541-555)polyclonal antibody, catalog No. EB09858 supplied by Everest Biotech(Upper Heyford, Oxfordshire UK). The capture agent recognizes aninternal sequence of protein ACO2, also known as mitochondrial aconitatehydratase. The entry number and the entry name for human ACO2 in theUniProt database are Q99798 and ACON HUMAN, respectively. The entrynumber and the entry name for mouse ACO2 in the UniProt database areQ99KI0 and ACON MOUSE, respectively. The sequence of an immunizingpeptide used for the antibody production is QDTYQHPPKDSSGQH (SEQ ID NO:8). Conjugation of the antibody to magnetic agarose beads andimmunoaffinity enrichment reactions were performed using proceduresdescribed in the previous Examples. The immunoaffinity enrichment wasperformed independently from two samples, namely trypsin-digestedlysates of cultured MKN-45 cells and trypsin-digested lysates of a mousebrain tissue. The digestive enzyme used for sample preparation wasbovine trypsin from Worthington Biochemical Corp (Lakewood, N.J.),catalog No. LS02119.

Mass spectra of proteolytic peptides captured from the digested MKN-45lysates exhibited a pair of prominent peaks between 4,400 and 4,800 m/zand another pair of peaks between 2,200 and 2,400 m/z. Using availableinformation about the protein sequence, the antibody specificity and thedigestive enzyme specificity, the pair of peaks observed in the higherm/z range was assigned to proteolytic peptidesLEAPDADELPKGEFDPGQDTYQHPPKDSSGQHVDVSPTSQR (SEQ ID NO: 9) andFRLEAPDADELPKGEFDPGQDTYQHPPKDSSGQHVDVSPTSQR (SEQ ID NO: 10), which havepredicted average m/z values of 4477.7 and 4781.1, respectively. Thepair of peaks observed in the lower m/z range was assigned to thedouble-charged forms of the same peptides.

Mass spectra of proteolytic peptides captured from the digested mousebrain tissue exhibited a pair of prominent peaks between 3,800 and 4,000m/z and a series of smaller peaks below 2,500 m/z. Using availableinformation about the protein sequence, the antibody specificity and thedigestive enzyme specificity, the pair of peaks observed between 3,800and 4,000 m/z was assigned to proteolytic peptidesFKLEAPDADELPRSDFDPGQDTYQHPPKDSSGQR (SEQ ID NO: 11) andKFKLEAPDADELPRSDFDPGQDTYQHPPKDSSGQR (SEQ ID NO: 12), which havepredicted average m/z values of 3846.1 and 3974.3, respectively. Two ofthe peaks observed in the lower m/z range were assigned to thedouble-charged forms of the same peptides.

In this Example, the antibody recognizes and specifically bindsproteolytic peptides derived from both human and mouse forms of ACO2because the epitope sequence is preserved in these proteins. Thedifferences in detected m/z values between the proteolytic peptides ofhuman and mouse origins are ascribed to a combination of two factors:(1) different recognition sites for the digestive enzyme, e.g. trypsinin the corresponding precursor proteins, which result in differentlength of the proteolytic peptides and (2) amino acid substitutions inthe corresponding precursor proteins, which do not alter recognitionsites for the digestive enzyme but nevertheless cause a mass difference.

This Example demonstrates a method of detection and quantification ofproteins derived from two different species, namely human (Homo sapiens)and mouse (Mus musculus). One application of this technology is asingle-plex or multiplex analysis of protein expression and/or proteinmodification in cell, tissue and organ transplants, e.g. tumorxenografts.

Example 15 Microarray Reactive Site Configured for Capturing ProteolyticPeptides Produced by Different Digestive Enzymes

In this Example the capture agent is Phospho-4E-BP1 (Thr37/46) (236B4)Rabbit monoclonal antibody, catalog No. 2855 purchased from CellSignaling Technology. According to the description provided on themanufacturer's website, which was accessed on Feb. 20, 2017, #2855antibody “ . . . detects endogenous levels of 4E-BP1 only whenphosphorylated at Thr37 and/or Thr46. This antibody may cross-react with4E-BP2 and 4E-BP3 when phosphorylated at equivalent sites”. The entrynumber and the entry name for human Eukaryotic translation initiationfactor 4E-binding protein 1 in the UniProt database are Q13541 and4EBP1_HUMAN, respectively. Conjugation of the antibody to magneticagarose beads and immunoaffinity enrichment reactions were performedusing procedures described in the previous Examples. The immunoaffinityenrichment of proteolytic peptides containing the antibody recognitionsite was performed from several samples, which were prepared byenzymatic digestion of MKN-45 cell lysates using the following digestiveenzymes: sequencing grade modified trypsin (Promega catalog No. V5117),sequencing grade chymotrypsin (Promega catalog No. V1061), pepsin(Promega catalog No. V1959), MS grade Lys-C protease (ThermoFishercatalog No. 90051), MS grade Lys-N protease (ThermoFisher catalog No.90300), MS grade Glu-C protease (ThermoFisher catalog No. 90054),sequencing grade Arg-C protease (Promega catalog No. V1881), thermolysin(Promega catalog No. V4001), elastase (Promega catalog No. V1891).

Mass spectrometric analysis of proteolytic peptides eluted from thebeads after the immunoaffinity enrichment was performed in the linearpositive mode as described in the previous Examples. Positions of peaksobserved in the mass spectra were in agreement with the m/z valuespredicted for the epitope-containing fragments of 4E-BP1 after digestionwith a specific enzyme. For example, two strong peaks in the massspectrum of trypsin-digested 4E-BP1 were observed near m/z 3129.4 and3209.3 corresponding to the calculated average m/z values of single- anddouble-phosphorylated forms of the peptideVVLGDGVQLPPGDYSTTPGGTLFSTTPGGTR (SEQ ID NO: 3) containing amino acids 21through 51 of human 4E-BP1, respectively. Likewise, the mass spectrum ofpepsin-digested 4E-BP1 exhibited a strong peak near m/z of 1393.5corresponding to a single-phosphorylated form of the peptideFSTTPGGTRIIY (SEQ ID NO: 13) containing amino acids 43 through 54 ofhuman 4E-BP1. The mass spectrum of chymotrypsin-digested 4E-BP1exhibited a pair of strong peaks near m/z of 1246.3 and 2053.3. Theformer peak was assigned to a single-phosphorylated form of the peptideSTTPGGTRIIY (SEQ ID NO: 14) containing amino acids 44 through 54 ofhuman 4E-BP1 and zero missed cleavages by chymotrypsin while the latterpeak was assigned to a single-phosphorylated form of the peptideFSTTPGGTRIIYDRKFL (SEQ ID NO: 15) containing amino acids 43 through 59of human 4E-BP1 and three missed cleavages by chymotrypsin.

The pepsin-digested samples also contain peptide ST[pT]PGGTL (SEQ ID NO:16) (0 missed cleavages, MW 813.78), which is recognized by #2855antibody. This peptide is derived from two proteins: 4E-BP1 and 4E-BP3(UniProt entry number 060516). By contrast, the pepsin-digested peptideGGEESQFEMDI (SEQ ID NO: 17) (1 missed cleavage, MW 1242.31), which isrecognized by the previously described #9644 antibody, is derived from4E-BP1 but not 4E-BP3. Thus including both #2855 and #9644 antibodiescreates a microarray, in which one capture agent recognizes an epitopethat is found in two distinct proteins and the other capture agentrecognizes an epitope that is found in only one of these proteins.

In a separate experiment, a reactive site containing the previouslydescribed antibody #4858 was added to the microarray. Both the #2855 and#4858 antibodies recognize epitopes, which lack proteolytic cleavagesites recognized by at least two of the following digestive enzymes:trypsin, chymotrypsin, pepsin, endoproteinase Glu-C and endoproteinaseAsp-N.

Example 16 Non-Specific Binding of a Target Analyte to Distinct ReactiveSites of a Microarray

Several identical microarrays featuring distinct reactive sites wereproduced using three phospho-specific antibodies #2855, #3077 and #4858described in the previous Examples. Less than ten (three to five)replicate beads containing the same type of antibody were included ineach microarray. Each microarray thus contained a total of nine reactivesites capable of binding proteolytic peptides derived fromphospho-4E-BP1 (Thr37/46), phospho-Met (Tyr1234/1235) and phospho-S6ribosomal protein (Ser235/236). The fabricated microarrays wereincubated with a series of digested MKN-45 cell lysates prepared usingthe enzymes listed in the previous Example, namely trypsin,chymotrypsin, Glu-C protease, Lys-C protease and Lys-N protease. Theimmunoaffinity enrichment from each of the digested cell lysates wasperformed as described in Example 6 except that 2.5M NaCl was omittedfrom the binding buffer during the binding reaction and the beads werewashed in 1×PBS buffer instead of the 2.5M solution of NaCl. Analytescaptured by the individual reactive sites of each microarray wereanalyzed by mass spectrometry as previously described. The absence of2.5M NaCl in the binding and wash buffers did not have a significanteffect on the mass spectra of analytes captured from trypsin-digestedcell lysates. In contrast, not using high salt in the binding and washbuffers during immunoaffinity enrichment performed fromchymotrypsin-digested cell lysates had a significant effect on the massspectra with a strong peak observed near m/z of 2222.5 in the spectrarecorded from every reactive site irrespectively of the conjugatedantibody. The non-specific 2222.5 peak was present in the mass spectrain addition to the analyte-specific peaks, which matched the expectedm/z values of proteolytic fragments of the corresponding precursorproteins after chymotrypsin digestion. For example, mass spectrarecorded from the phospho-4E-BP1 reactive sites exhibited the 2222.5peak in addition to the 1246.3 and 2053.3 peaks identified in theprevious Example. To identify the non-specifically binding compoundresponsible for the 2222.5 peak an eluate was collected from bovineserum albumin (BSA)-conjugated agarose beads, which have been exposed toa chymotrypsin-digested MKN-45 cell lysate. The eluate was analyzed byEdman degradation yielding a peptide sequence KAQKKDGKKRKRSRKESY (SEQ IDNO: 18), which has a predicted average [M+H]+ value of 2222.6 andtentative assignment to amino acids 21 through 38 of human Histone H2Btype 1-C/E/F/G/I. The entry number and entry name for human Histone H2Btype 1-C/E/F/G/I in the UniProt database are P62807 and H2B1C HUMAN,respectively. The data was subsequently used to make an entry in themicroarray decoding table, which contained information about themolecular weight of this non-specifically binding compound and the factthat this compound may appear in the mass spectra of samples of humanorigin such as human cell cultures that have been subjected to digestionwith chymotrypsin.

Example 17 Specific Binding of a Target Analyte to Distinct ReactiveSites of a Microarray

Polyclonal antibodies used as capture agents were from Everest Biotech.These included: (1) goat anti-aconitase 2 antibody (catalog No. EB09857,immunogenic peptide sequence C-QHVDVSPTSQRLQ (SEQ ID NO: 19)); (2) goatanti-aconitase 2 (aa541-555) antibody (catalog No. EB09858, immunogenicpeptide sequence C-QDTYQHPPKDSSGQH (SEQ ID NO: 20)); (3) goatanti-GPI/Neuroleukin antibody (catalog No. EB09739, immunogenic peptidesequence C-YREHRSELNLRR (SEQ ID NO: 21)) and (4) goat anti-IDH3B(aa369-383) antibody (catalog No. EB10997, immunogenic peptide sequenceC-TTDFIKSVIGHLQTK (SEQ ID NO: 22))

Individual antibodies were conjugated to magnetic agarose beads usingprocedures described in the previous Examples. A 4-plex bead arraycontaining 8 reactive sites was assembled by selecting 2 beadsconjugated to one of the four antibodies listed above and combiningbeads conjugated to different antibodies in a single 1.7 mLmicrocentrifuge tube. The assembled microarray was subsequently reactedwith 2 mg of chymotrypsin digested MKN-45 cell lysate prepared asdescribed in the previous Examples. The reacted bead array wastransferred on a microwell slide, analytes eluted from the beads andanalyzed by MALDI TOF MS as described in the previous Examples.

A total of 8 mass spectra were identified that had one or several peakswith a signal-to-noise ratio greater than 3:1 in the 1,000-5,000 m/zmass range, matching the number of reactive sites present in themicroarray. Four distinct pairs of mass spectra were identifiedcorresponding to 4 distinct reactive sites of the microarray. Massspectra recorded from the replicate reactive sites were very similar,e.g. had the same number of peaks with m/z values that varied by lessthan 0.1 between different spectra. FIG. 7 shows exemplary mass spectraacquired from individual reactive sites. Table 1 shows a decoding tablefor this microarray. In this Example the same peptide target with asequence of QHPPKDSSGQHVDVSPTSQRL (SEQ ID NO: 23) and m/z of 2301.49 wascaptured by two distinct reactive sites of the microarray, namelyreactive sites containing antibodies EB09857 and EB09858. Theseantibodies recognize adjacent sequences within human aconitase 2, whichallows them to specifically bind the same proteolytic peptide. However,antibody EB09857 additionally captured a peptide with a sequence ofQHVDVSPTSQRLQLLEPFDKWDGKDLED (SEQ ID NO: 24) and an m/z of 3297.60,which was not captured by antibody EB09858. Therefore, as shown in Table1, the two reactive sites (ACO2 and ACO2 (541-555)) may be readilydistinguished within the microarray, as both bind a target with m/z of2301.49, yet only the former binds a target with m/z of 3297.60. Infact, the entry in the decoding table associated with the reactive siteACO2 (541-555) may optionally contain specific reference to the absenceof a signal at m/z of 3297.60, as depicted by the *SIGNAL ABSENT* notein Table 1. Furthermore, in reference to FIG. 7 it can be seen that boththe ACO2 and ACO2 (541-555) spectra also contain several additionallower intensity peaks. These peaks are specific to their respectiveantibodies and therefore information about their MW, m/z or TOF valuesmay be included in the decoding table to further increase confidence inthe target identification.

TABLE 1 Microarray decoding table for a 4-plex microarray Reactive SiteTarget ID SEQ ID NO: m/z ACO2 QHPPKDSSGQHVDVSPTSQRLQHVDVSPTSQ 23 2301.49RLQLLEPFDKWDGKDLED 24 3297.60 ACO2 (541-555) QHPPKDSSGQHVDVSPTSORL 232301.49 *SIGNAL ABSENT* 3297.60 GPI YREHRSEL 25 1090.19 IDH3STTTDFIKSVIGHLQTKGS 26 2021.29

A simplified version of a decoding table for the previously describedmicroarray is shown in Table 2. In this Example, an amino acid sequenceof a target captured by a reactive site is not provided and the reactivesite is identified solely on the basis of one or more signals detectedin a mass spectrum. Furthermore, a reactive site may be identified onthe basis of a signal, which is not detected at a specific m/z in a massspectrum, as shown for the reactive site ACO2 (541-555) in Table 2.

In this Example, the ACO2 reactive site is associated with a combinationthat includes 2 distinct values: 2301.49 and 3297.60. The combination isboth necessary and sufficient for identifying a target that specificallybinds to the capture agent of that reactive site.

TABLE 2 Simplified microarray decoding table for a 4-plex microarrayReactive Site Signal Detected in a Mass Spectrum (m/z) ACO2 2301.49 AND3297.60 ACO2 (541-555) 2301.49 AND NOT 3297.60 GPI 1090.19 IDH3 2021.29

In some experiments, two goat anti-GAPDH (internal) antibodies fromEverest Biotech were added to the microarray: catalog No. EB07069,immunogenic peptide sequence C-GVNHEKYDNSLK (SEQ ID NO: 27) and catalogNo. EB06377, immunogenic peptide sequence C-HQVVSSDFNSDT (SEQ ID NO:28). The epitope recognized by the latter antibody lacks a proteolyticcleavage site recognized by trypsin, while the epitope recognized by theformer antibody contains such site, i.e. an internal lysine.

Example 18 Providing an Estimate of Relative Abundance of a TargetAnalyte in a Sample

A microarray was produced as described in the previous Examples usingpreviously described antibodies #4858 and #9644, as well asphospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbitmonoclonal antibody, catalog No. 4370 from Cell Signaling Technology.According to the description provided on the manufacturer's website,which was accessed on Mar. 25, 2017, #4370 antibody “ . . . detectsendogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when duallyphosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2),and singly phosphorylated at Thr202”.

It was experimentally determined that each of the 3 antibodies listedabove was capable of specifically binding several targets from trypsindigested MKN-45 cell lysates. It was also experimentally determined thatthe relative intensity of signals from multiple targets captured by thesame antibody did not vary considerably between different samplepreparations, as long as cell culture and sample preparation conditionsremained similar, e.g. same cell culture medium and same digestiveenzyme were used. For example, the intensity ratio of peaks recordedfrom proteolytic fragments RAGGEESQFEMDI (SEQ ID NO: 1) and AGGEESQFEMDI(SEQ ID NO: 2) of 4E-BP1, which have m/z of 1469.6 and 1313.4,respectively, was consistently greater than 10:1. Peaks from the doubly-and triply-phosphorylated forms of the peptide RLSSLRASTSKSESSQK (SEQ IDNO: 7) were about 2-fold more intense than peaks from the peptideRLSSLRASTSKSESSQK (SEQ ID NO: 7) that contained 4 or 5 phosphates, whichin turn were about 3-fold more intense than peaks from the same peptidecontaining 6 or 7 phosphates. Signal from VADPDHDHTGFL[pT]E[pY]VATR (SEQID NO: 29) (Erk2) near m/z of 2305.3 was 5- to 20-fold stronger comparedto signal from IADPEHDHTGFL[pT]E[pY]VATR (SEQ ID NO: 30) (Erk1) near m/zof 2333.3.

Accordingly, as shown in Table 3, it is possible to include the relativepeak intensity data in the microarray decoding table, which in this caseis given for a specific cell line (e.g. MKN-45), specific treatmentconditions (e.g. DMSO treatment or a kinase inhibitor treatment), andspecific digestion conditions (e.g. mass spec sequencing grade trypsin).Relative intensity of peaks in a mass spectrum is often directly relatedto relative amounts of the corresponding analytes in a sample andproviding such information in the decoding table may assist in theidentification of the target analytes. Specifically, data in Table 3indicates that a proteolytic fragment of Erk2 that is doublyphosphorylated at Thr185 and Tyr187 is more abundant in trypsin-digestedMKN-45 cell lysates than a proteolytic fragment of Erk1 that is doublyphosphorylated at Thr202 and Tyr204. In some cases, the relationshipbetween the intensity of a signal in a mass spectrum and the analyteabundance in a sample is more complex, e.g. for peptides with variablenumbers of PTM sites. Nevertheless, providing a spectral pattern thatincludes abundance data in a microarray decoding table is a usefulmethod of identifying the target. For example, while both Phospho-S6Ribosomal Protein (Ser235/236) and Phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204) reactive sites are able to specifically bind a targetwith a molecular weight near 2333 Da, the microarray decoding tablecontaining abundance information can help provide unambiguous assignmentof the detected target analyte. In this Example, two targets recognizedby distinct capture agents of the microarray have molecular weights thatdiffer by less than 1 Da (2332.9 and 2333.3), while molecular weights ofother targets recognized by the capture agents are separated by at least5 Da to ensure unambiguous assignment.

TABLE 3Microarray decoding table including an estimate of relative target abundanceReactive Site Target Sequence SEQ ID NO: m/z Comments 4E-BP1 totalAGGEESQFEMDI  2 1313.4 1469 peak RAGGEESQFEMDI  1 1469.6is about 10-fold stronger than 1313 peak Phospho-S6RLSSLRASTSKSESSQK: 2P  7 2013.1 2013 and 2093 peaks Ribosomal ProteinRLSSLRASTSKSESSQK: 3P  7 2093.0 have similar intensity (Ser235/236)RLSSLRASTSKSESSQK: 4P  7 2173.0 and are about 2-foldRLSSLRASTSKSESSQK: 5P  7 2252.9 stronger that 2173 andRLSSLRASTSKSESSQK: 6P  7 2332.9 2252 peaks. 2173 andRLSSLRASTSKSESSQK: 7P  7 2412.9 2252 peaks are about3-fold stronger that 2332 and 2412 peaks Phospho-p44/42VADPDHDHTGFLT*EY*VATR 29 2305.3 2305 peak is about 5 MAPKIADPEHDHTGFLT*EY*VATR 30 2333.3 to 20-fold stronger (Erk1/2)than 2333 peak (Thr202/Tyr204)

Example 19 Microarray Configured for Measuring PTM Status of MultipleSites within a Protein

The target analyte is human S6 ribosomal protein, also known asribosomal protein S6 (rpS6, UniProt entry number P62753) that wasdescribed in the previous Examples. According to the online database ofprotein post-translational modifications (PhosphoSitePlus®), rpS6 may bephosphorylated at multiple sites including a cluster of residues locatedat the protein C-terminus: Ser235, Ser236, Ser240, Ser242, Ser244,Ser246, Ser247.

A microarray for measuring phosphorylation status of individual siteswithin rpS6 was produced using previously described methods. Themicroarray included three different antibodies, which were individuallyconjugated to their respective beads: (1) the previously describedmonoclonal rabbit antibody, catalog No. 4858 from Cell SignalingTechnology; (2) a polyclonal rabbit antibody, catalog No. E-AB-32812from Elabscience (Houston, Tex.); (3) a polyclonal rabbit antibody,catalog No. E-AB-32813, also from Elabscience. According to the productdescription available on Elabscience's website, the immunogen forE-AB-32812 is a synthetic peptide derived from human rpS6 “around thenon-phosphorylation site of Ser235”, while the immunogen for E-AB-32813is a synthetic peptide derived from human rpS6 “around thenon-phosphorylation site of Ser240”. The reactive site that containsantibody #4858 is expected to bind proteolytic fragments of rpS6 thatare dually phosphorylated at Ser235/Ser236, as well as proteolyticfragments phosphorylated at Ser240, Ser242, Ser244, Ser246 and/or Ser247in addition to Ser235/Ser236. Therefore, the epitope recognized by thecapture reagent of this reactive site contains two PTMs while some ofthe targets that bind to the reactive site contain one or moreadditional PTMs, which are located outside of the epitope. The reactivesite that contains antibody #E-AB-32812 is expected to bind proteolyticfragments of rpS6 containing non-phosphorylated Ser 235. The reactivesite that contains antibody #E-AB-32813 is expected to bind proteolyticfragments of rpS6 containing non-phosphorylated Ser 240. Because rpS6may be phosphorylated at several distinct sites, each of the microarrayreactive sites is expected to bind proteolytic peptides that havedifferent molecular weights.

The reactive sites of the fabricated microarray were each reacted with200 μg of trypsin-digested lysate of MKN-45 cells, which was prepared aspreviously described. In this Example, each of the microarray reactivesites was individually incubated with the digested lysate andsubsequently prepared for MS analysis. Accordingly, the identity of anantibody conjugated to a particular reactive site was known prior to andduring the MS analysis. MALDI TOF mass spectra were acquired fromindividual reactive sites as previously described. The mass spectra ofproteolytic peptides captured by the antibody #4858 displayed severalprominent peaks near average m/z of 2013.1, 2093.0, 2173.0, 2252.9,2332.9 and 2412.9, which were assigned to C-terminal fragments of rpS6(amino acids 233 through 249) containing 2, 3, 4, 5, 6 and 7phosphorylated sites, respectively. The mass spectra of proteolyticpeptides captured by the antibody E-AB-32813 displayed additional peaksnear m/z of 1853.1 and 1933.0, which were assigned to C-terminalfragments of rpS6 (amino acids 233 through 249) containing 0 and 1phosphorylated site, respectively. The 1853.1 and 1933.0 peaks were notdetected in the mass spectra obtained using the antibody #4858, whichrequires a presence of at least 2 phosphorylated sites. Likewise, the2252.9, 2332.9 and 2412.9 peaks were not detected in the mass spectraobtained using the antibody E-AB-32813, which requires a presence of atleast one and possibly more non-phosphorylated sites. The mass spectraobtained using the antibody E-AB-32812 displayed a weak peak near m/z of1853.1 corresponding to the non-phosphorylated C-terminal fragment ofrpS6 and no detectable peaks at higher m/z. The low intensity of thissignal may be due to the more efficient trypsin digestion ofnon-phosphorylated rpS6, which contains several internal Arg and Lysresidues, compared to its phosphorylated counterpart. This Example showsa microarray that contains distinct capture agents that specificallyrecognize distinct epitopes in a sequence of a naturally occurringprotein, with distinct capture agents being associated with distinctbeads. It also shows a microarray that contains distinct capture agentsthat specifically recognize a protein site (Ser235 in rpS6) in theabsence and in the presence of a PTM, in this case phosphorylation.

Example 20 Microarray for Measuring Multiple Proteolytic Fragments of aProtein

The protein is the previously described human eukaryotic translationinitiation factor 4E-binding protein 1 (4E-BP1, UniProt entry numberQ13541). According to the PhosphoSitePlus® database, 4E-BP1 may bephosphorylated at multiple sites including Thr37, Thr46, Ser65, Thr70,Ser101 and Ser112.

A microarray for measuring phosphorylation status of individual siteswithin 4E-BP1 was produced using the previously described methods. Themicroarray included 4 antibodies, all purchased from Cell SignalingTechnology, which were individually conjugated to their respectivebeads: (1) phospho-4E-BP1 (Thr37/46) (236B4) rabbit mAb (catalog No.2855); (2) phospho-4E-BP1 (Ser65) (D9G1Q) rabbit mAb (catalog No.13443); (3) phospho-4E-BP1 (Thr70) (D7F61) rabbit mAb (catalog No.13396) and (4) 4E-BP1 (53H11) rabbit mAb (catalog No. 9644). The first 3antibodies on this list are designed to recognize correspondingphosphorylated sites of human 4E-BP1 while the #9644 antibody isdesigned to probe the total amount of 4E-BP1. Thus, the microarraycontained 4 distinct reactive sites that were able to probe differentsites within 4E-BP1. The microarray contained fewer than 5 replicates ofeach of the 4 distinct reactive sites.

According to the product description on the manufacturer's website, the#9644 antibody “is produced by immunizing rabbits with a syntheticpeptide corresponding to residues surrounding Ser112 of human 4E-BP1”.It was experimentally verified that the reactive site containing thisantibody efficiently captured a synthetic peptide containing sequenceRAGGEESQFEMDI (SEQ ID NO: 1) but did not bind a peptide containingsequence RAGGEE[pS]QFEMDI (SEQ ID NO: 31), in which residuecorresponding to Ser112 of 4E-BP1 was phosphorylated. Thus, the #9644antibody specifically recognized a non-phosphorylated peptide in apresence of a phospho peptide containing the same amino acid sequence.

The fabricated microarray was incubated with 200 μg of trypsin-digestedlysate of MKN-45 cells, which was prepared as previously described. Thepeptides captured on individual reactive sites were measured by linearand reflector MALDI TOF MS and also subjected to MS-MS sequencing usingthe LIFT mode of Bruker Autoflex. After the sequences of peptidescaptured on individual reactive sites were determined by LIFT, it waspossible to assign them to each of the 4 antibodies included in thearray, based on the antibody specificity. The reactive site containing#9644 antibody captured peptides RAGGEESQFEMDI (SEQ ID NO: 1) (1 missedcleavage, 1\4W 1469.57) and AGGEESQFEMDI (SEQ ID NO: 2) (0 missedcleavages, MW 1313.39). The reactive site containing #2855 antibodycaptured peptides VVLGDGVQLPPGDYST[pT]PGGTLFSTTPGGTR (SEQ ID NO: 32) (0missed cleavages, MW 3129.36), VVLGDGVQLPPGDYSTTPGGTLFST[pT]PGGTR (SEQID NO: 33) (0 missed cleavages, MW 3129.36) andVVLGDGVQLPPGDYST[pT]PGGTLFST[pT]PGGTR (SEQ ID NO: 34) (0 missedcleavages, MW 3209.3449). In addition, peptides containing 1 and 2missed cleavage sites such as VVLGDGVQLPPGDYST[pT]PGGTLFSTTPGGTRIIYDRK(SEQ ID NO: 35) were also captured. The reactive site containing #13443antibody captured peptides FLME[camC]RN[pS]PVTKTPPR (SEQ ID NO: 36) (2missed cleavage sites, MW 2014.28) andFLME[camC]RN[pS]PVTKTPPRDLPTIPGVTSPSSDEPPMEASQSHLR (SEQ ID NO: 37) (3missed cleavage sites, MW 4745.29). The reactive site containing #13396antibody captured peptides FLME[camC]RNSPVTK[pT]PPR (SEQ ID NO: 38) (2missed cleavage sites, MW 2014.28) andFLME[camC]RNSPVTK[pT]PPRDLPTIPGVTSPSSDEPPMEASQSHLR (SEQ ID NO: 39) (3missed cleavage sites, MW 4745.29). [camC] denotes carbamidomethylcysteine. All peptides had natural isotope abundance.

In this Example, the microarray contained distinct capture agents,namely antibodies #13443 and #13396, which were associated with distinctbeads and which individually recognized distinct epitopes containingphospho-Ser65 and phospho-Thr70, respectively, within a fragment ofhuman 4E-BP1 that has a molecular weight less than 5000 Da. The site ofthe first PTM (phospho-Ser65) is separated from the site of the secondPTM (phospho-Thr70) by less than 10 amino acids.

In this Example, the epitope recognized by the antibody #2855, namelythe ST[pT]P sequence (SEQ ID NO: 40) occurs naturally in both human andmouse 4E-BP1.

In this Example, the epitopes recognized by the antibodies #2855 and#9644 lack proteolytic cleavage sites that are recognized by trypsin andchymotrypsin.

In this Example, three distinct PTM-containing fragments of a proteinwere captured on distinct reactive sites of the microarray.

In this Example, the antibodies used for capturing proteolytic peptideshave been validated by the manufacturer for assays including westernblot, immunoprecipitation, immunofluorescence, immunohistochemistry,flow cytometry and ELISA.

The sequence length of 4E-BP1 is 118 amino acids. The length ofproteolytic fragments of 4E-BP1, which were captured by individualreactive sites of the microarray, ranged from 13 amino acids to 42 aminoacids. The combined sequence length of all non-overlapping proteolyticfragments that were captured by the microarray was 86 amino acids, morethan 70% of the sequence length of the protein. In this Example, it waspossible to probe a substantial portion of a protein, e.g. more than 50%of a sequence length of a protein using a microarray containing fewerthan 10 distinct capture agents.

In a separate experiment, the same microarray was reacted with achymotrypsin-digested lysate of MKN-45 cells. The reactive sitecontaining antibody #9644 captured peptides RNSPEDKRAGGEESQFEMDI (SEQ IDNO: 41) (1 missed cleavage site, MW 2296.44) and RN[pS]PEDKRAGGEESQFEMDI(SEQ ID NO: 42) (1 missed cleavage site, MW 2376.42). The epitoperecognized by the capture agent of this reactive site did not contain aPTM, while one of the captured peptide targets contained the PTM,namely, phosphorylation, in a position corresponding to Ser101 of human4E-BP1.

Example 21 Microarray Reactive Site Configured for Binding a TargetContaining Different PTM Types

R&D Systems (Minneapolis, Minn.) Human/Mouse/Rat Phospho-CDC2/CDK1 (Y15)antibody, catalog No. AF888 recognizes a chymotrypsin-digested fragmentof human cyclin-dependent kinase 2 (UniProt entry number P24941)[Ac]MENFQKVEKIGEGT[pY]GVVY (SEQ ID NO: 43) (2 missed cleavages, MW2314.52), which contains both acetylation at the N-terminus andphosphorylation of the amino acid corresponding to Tyr15 of human CDC2.The first PTM (phosphorylation) is located within the epitope, while thesecond PTM (acetylation) is located outside of the epitope. In thisExample, the site of the first PTM is separated from the site of thesecond PTM by more than 10 amino acids within a peptide that has amolecular weight less than 4000 Da.

Example 22 Capture Agent Configured for Binding Targets Derived fromDistinct Proteins that are Constituents of Distinct Biological Pathways

An antibody that recognizes an amino acid sequence PKEAP (SEQ ID NO:44), which is found in human STAT1 (UniProt entry number P42224), alsorecognizes an amino acid sequence PKPAP (SEQ ID NO: 45), which is foundin transcription factor p65 (UniProt entry number Q04206). The formerprotein is signal transducer and transcription activator while thelatter is a transcription factor. MALDI MS—detectable fragments of STAT1and p65 containing these sequences can be produced by chymotrypsindigestion of their respective precursor proteins in lysates of MKN-45cells using previously described methods. Such antibody is availablefrom Cell Signaling Technology as Statl (D1K9Y) Rabbit mAb, catalognumber #14994.

Example 23 Microarray Configured for Binding Intact Protein and SmallMolecule Target Analytes

The microarray included antibodies that have been validated by theirrespective manufacturers to recognize intact, i.e. non-digested,proteins and small molecule targets. Mouse monoclonal antibodyrecognizing human insulin (MW 5807.6 Da) was from Novus Biologicals,catalog number NBP2-32975. Rabbit polyclonal antibody recognizingfull-length human C Reactive Protein (MW 25039 Da) was from Abcam,product code ab31156. Mouse monoclonal antibody recognizing cortisol (MW362.46) was from Abcam, product code ab116600. Mouse monoclonal antibodyrecognizing testosterone (MW 288.43) was from Novus Biologicals, catalognumber NBP1-78562.

The microarray contained antibodies that recognize small molecules withMW less than 700 Da, as well as antibodies that recognize intactproteins with MW greater than 5000 Da and 10000 Da (insulin and CRP,respectively).

The microarray also contained beads conjugated to Protein A+G. In theabsence of cross-linking, the antibodies listed in this Example wereeluted from the Protein A+G conjugated beads and detected by MALDI TOFMS using sinapinic acid as the matrix. The antibodies were detected inthe molecular weight range between approximately 130 kDa and 170 kDa,depending on the specific antibody sequence. This Example shows thatProtein A+G may serve as the capture reagent for binding proteins, e.g.antibodies that have molecular weight greater than 100 kDa.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thepresent disclosure has been described in connection with the specificembodiments thereof, it will be understood that it is capable of furthermodification. Furthermore, this application is intended to cover anyvariations, uses, or adaptations of the disclosure, including suchdepartures from the present disclosure as come within known or customarypractice in the art to which the disclosure pertains, and as fall withinthe scope of the appended claims.

What is claimed is:
 1. A method for supplying a bead array system thatis intended for analyzing a sample, the method comprising: providing abead array, the bead array comprising a first reactive site and a secondreactive site, the first reactive site comprising a first bead and afirst capture agent, wherein the first capture agent is configured tospecifically bind a first target and a second target, a molecular weightof the first target being different from a molecular weight of thesecond target, the second reactive site comprising a second bead and asecond capture agent, the second capture agent being distinct from thefirst capture agent, wherein the second capture agent is configured tospecifically bind a third target and providing a decoding table, thedecoding table containing information that is obtained prior toanalyzing the sample, said information comprising values that arederived from the molecular weight of the first target, the molecularweight of the second target and the molecular weight of the thirdtarget.
 2. The method of claim 1 wherein the first, the second and thethird targets are distinct proteolytic fragments of a naturallyoccurring protein.
 3. The method of claim 1 wherein the first, thesecond and the third targets are polypeptides that contain distinctamino acid modifications within an otherwise identical amino acidsequence.
 4. The method of claim 1 wherein the first capture agent isone of a monoclonal antibody, a polyclonal antibody, an antibodyfragment, a single domain antibody, an aptamer, a protein, apolypeptide, a receptor, a ligand, an enzyme, an enzyme substrate and anenzyme inhibitor.
 5. The method of claim 1 wherein the decoding tablefurther contains an entry that is associated with the first reactivesite, the entry containing information about a target that does notspecifically bind to the first reactive site.
 6. The method of claim 1wherein the decoding table is provided electronically.
 7. The method ofclaim 1 wherein the decoding table information is generated by acomputer algorithm.
 8. The method of claim 1 wherein the decoding tablefurther contains a sequence information that is obtained by massspectrometry.
 9. The method of claim 1 wherein the decoding tablefurther contains estimated abundances of the first target and the secondtarget in the sample.
 10. A method for using a bead array system, themethod comprising: contacting a sample with a bead array, the bead arraycomprising a first reactive site and a second reactive site, the firstreactive site comprising a first bead and a first capture agent, whereinthe first capture agent is configured to specifically bind a firsttarget and a second target, a molecular weight of the first target beingdifferent from a molecular weight of the second target, the secondreactive site comprising a second bead and a second capture agent, thesecond capture agent being distinct from the first capture agent,wherein the second capture agent is configured to specifically bind athird target, receiving a decoding table, the decoding table containinginformation that is obtained prior to analyzing the sample, saidinformation comprising values that are derived from the molecular weightof the first target, the molecular weight of the second target and themolecular weight of the third target, after the contacting step,analyzing the bead array using mass spectrometry, detecting a signal ina mass spectrum and identifying a target analyte that is associated withthe signal by verifying that an m/z value of the signal or an equivalentthereof is present in the decoding table.
 11. The method of claim 10wherein the first, the second and the third targets are distinctproteolytic fragments of a naturally occurring protein.
 12. The methodof claim 10 wherein the first, the second and the third targets arepolypeptides that contain distinct amino acid modifications within anotherwise identical amino acid sequence.
 13. The method of claim 10wherein the first capture agent is one of a monoclonal antibody, apolyclonal antibody, an antibody fragment, a single domain antibody, anaptamer, a protein, a polypeptide, a receptor, a ligand, an enzyme, anenzyme substrate and an enzyme inhibitor.
 14. The method of claim 10wherein the decoding table further contains an entry that is associatedwith the first reactive site, the entry containing information about atarget that does not specifically bind to the first reactive site. 15.The method of claim 10 wherein the decoding table is receivedelectronically.
 16. The method of claim 10 wherein the decoding tableinformation is generated by a computer algorithm.
 17. The method ofclaim 10 wherein the decoding table further contains a sequenceinformation that is obtained by mass spectrometry.
 18. The method ofclaim 10 wherein the decoding table further contains estimatedabundances of the first target and the second target in the sample.